REVERSE TRANSCRIPTASE

Authors: A. M.Wu
Litton Bionetics
Bethesda, Maryland
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R. C. Gallo
National Institutes of Health
Bethesda, Maryland

Referee: J . Schlom
National Institutes of Health
Bethesda, Maryland
For personal use only.

I. INTRODUCTION paved the way for the finding of viral-related

components, reverse transcriptase5-" and
A. Discovery type-C viral related nucleic acids in human leukemic
Recognition of RNA tumor viruses as causative cells by Gallo, Spiegelman, and their co-workers
agents of malignant disease started with the '
(see later section' O '-I ).
9'

observation (by Ellerman and Bang in 1908) that a

filterable agent transmitted avian leukemia' and In the late 1950s and early 1960s progress in
the subsequent isolation of chicken sarcoma virus the molecular biology of bacteria and the bacterio-
by Rous in 1911.' These findings were followed phages clearly showed that genetic information
by an intense and continuous search for new was transmitted from DNA to RNA and that
isolates, the establishment of inbred strains of genetic information was conserved in DNA. Due to
mice, and the development of tissue culture its predictability, studies on DNA viruses such as
techniques. In the early 1960s, the significance of polyoma and SV40 quickly benefited from the
RNA tumor viruses was generally accepted, knowledge gained from DNA phages. At this time,
especially in avian and murine systems, but with studies on RNA tumor viruses had just turned
some skepticism, probably because its role in from an emphasis on animal experiments to tissue
natural disease was not yet evident. Moreover, the culture. Investigations into the question of how an
mode of replication of the RNA tumor viruses was RNA tumor virus replicates and completes its life
still not understood. In recent years, the study of cycle was timely. Most studies were performed
these viruses markedly intensified, principally with avian leukosis sarcoma viruses and with
murine leukemia-sarcoma viruses.
because of a discovery which filled a major gap in
the understanding of their replication. This is the Generally, cells infected by RNA tumor viruses
independent discovery of the viral RNAdependent are not killed and the viral genetic information is
DNA polymerase (reverse transcriptase) by Temin transmitted to daughter cells at mitosis. The
and Mizutani' and by Baltimore4 in 1970. This structure that transmits virus information is called
discovery also extended our understanding of the the provirus. The first indication that DNA was
mode of genetic information transfer, and it involved in the reproduction of these viruses came

December 1975 289

 from some studies with inhibitors. It was observed double-stranded RNA was an intermediate in viral
that virus production was drastically reduced when replication was not completely ruled out. In fact,
infected cells were exposed to DNA synthesis attempts were made t o find an RNA+RNA
i n h i b i t o r s s u c h as actinomycin D, 5- replicase in cells infected by an RNA tumor
bromodeoxyuridine, 5-iododeoxyuridine, or cyto- virus.''
sine arabinoside early after infection.' '-'' In
In the late 1960s, following the isolation of
fact, Temin demonstrated that in chicken fibro-
sigma factor from purified Escherichia coli RNA
blasts transformed by Rous sarcoma virus, the
polymerase by Burgess and his colleagues3' and
provirus was a new DNA homologous to the
the finding of the induction of viral specific
RNA isolated from RSV as judged by molecular
hybridization.' ' These preliminary findings led
replicase in QP phage-infected cells,3' many
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Temin to propose a DNA provirus hypothesis' ' scientists began searching for nucleotide poly-
merases both in cells and in virions. The purpose
in 1964. The hypothesis stated that the provirus
was to understand gene regulation through elucida-
is DNA and that the replication of leukovirus
tion of the specific transcription of the viral
involved successive transfers of information genome. At that time, vaccinia virus, a DNA virus,
from viral RNA to proviral DNA to progeny viral was found to contain a DNA-dependent RNA
RNA, whereas the replication of the provirus p ~ l y m e r a s e , ' ~ Reovirus, a double-stranded
'9
accompanying cell multiplication involved in- RNA virus, an RNA-dependent RNA poly-
formation transfer from DNA to DNA. This merase: ,' and vesicular stomatitis virus, a
hypothesis could explain most of the findings single-stranded RNA virus, an RNAdependent
of Temin,' ' Bader,' and others, and it gained RNA polymerase?' With these findings it was
additional support from further studies. For logical to consider the existence of a replication
example, Baluda and Nayak observed sensitivity enzyme in RNA tumor viruses. In fact, some
of viral replication to actinomycin D and found other biological experiments provided an addi-
a new DNA sequence homologous to viral RNA tional hint. It was found that the exposure of
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in the viral transformed cell^.'^ The newly syn- stationary cells to protein synthesis inhibitors
thesized DNA was shown to be different from such as puromycin or cycloheximide did not affect
that synthesized in S-phase of the cell cycle.' 3' the formation of the provirus after infection by
It was observed that stationary cells exposed RSV.' *4 This finding suggested that the enzyme
to RSV were stably infected, but virus production
required for the formation of proviral DNA was
started only after cell division was initiated.
already present, i.e., it did not require protein
While the stationary cells are generally resistant
synthesis. Finally, the viral RNAdependent DNA
to DNA synthesis inhibitors, the establishment
polymerase was found in the virions of Rous
of a stable viral infection in these stationary
sarcoma virus by Temin and Mizutani' and
cells is sensitive to the same DNA synthesis in-
additionally in Rauscher leukemia virus by
hibitors. Furthermore, Balducci and Morgan'
Baltimore: findings quickly confirmed and
and Boettiger and Temin" showed that a stable
extended to a number of other viruses by several
infection of RSV was obtained by infecting
groups (e.g., References 41 and 42).
stationary chicken fibroblasts in the presence of
5'-bromodeoxyuridine. This infection is aborted
by exposing the infected cells to light and this B. After effects
effect is dependent on the multiplicity of RNAdependent DNA polymerases were soon
infection. These results suggested that a found in many RNA-containing viruses. At present
bromodeoxyuridine-containing DNA with infor- all of the known infectious t y p e 4 virus, type-B
mation required for the successful infection was virus, Mason-Pfizer monkey type virus, sheep slow
synthesized in the infected cells and this DNA was virus, and foamy virus have been shown to contain
a copy of the information from the input virus this DNA polymerase. These are listed in Table 1.
rather than a copy of cellular DNA. Since the procedure for assay of RNAdependent
With this convincing biological data, some DNA polymerases is relatively simple and the
Investigators (in addition to Temin) predicted a enzyme activity is relatively specific, this enzyme
possible existence of a DNA polymerase which has become a useful tool for many purposes, e.g.,
could transfer viral genetic information from for quantitating known virus, for studying the
RNA to DNA.29 9'' However, the possibility that process of viral replication, for searching for the
290 CRC Critical Reviews in Biochemistry
 TABLE 1

Particles Containing Reverse Transcriptase*

I. Extracellular Particles
A. Type€ particles
1. Leukemia-sarcoma viruses
1) Mammalian leukemia-sarcoma virus
Primate: SiSV, SiLV;' ' GaLV-1;' ' GaLV (SEATO);'
3' GBrl, GBr2, G B I ~'.O ~
Rodent: M-MuLV, M-MuSV(M-MuLV)f '' 3 K-MuLV, K-MuSV(K-MuLV);' AKR MuLV;' '
F-MuLV? ' R-MuLV;' *' ' H-MuSV;~' RaLV mister-Furth);' RaLV (MSV-O helper);' I
RaLV (R-35);' ' HaLV' ' and HaSV.'
Carnivore: GFeLV;' ' R-FeLV;' ' T-FeLV;' G-FeSV;' ' R-FeSV;' FS-1 virus.'
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Ungulate: Porcine type€ particles;' ' ' Bovine type-C particles.' ,5 '
9'

2) Avian leukosis-sarcoma virus

MC29(ALV);" AMV;' '"'B-77;"" RSV(RAV-1);' PR-RSV;' SR-RSV.3 9''

3) Avian REV.' p6 '

4) Reptile viper virus' ' * 2 '
2. Endogenous virus
Nonhuman primate: Baboon placenta virus (M-7).6
Rodent: Mouse ecotropic viruses (N and B tropic);6 3-6 ' Mouse xenotropic virus;6 6 - 7 $3
9' *
Rat endogenous virus;"-" Guinea pig endogenous virus;' v3 '
i76
Hamster endogenous
virus. ' '
Carnivore: RD114 virus;" CCC virus."
3. Particles isolated from human cells
E s p l ;' HL23 cells;' p4 ' *' '
Some human leukemic cells;' ' ' ' and normal human fibroblast strains,' ' and human carcinoma cell
9' 1'

line.' a '
B. Type-Bvirus
734 B virus;" MMTV (C3H) " and (Paris RlII);' ' Murine milk particle (C3H)' and (Paris III);' a Human milk
For personal use only.

particle.'O
C. MPMV' ' and MPMV-like virus
HeLa cells;' ' H e p 2 cells;' HBT-3 cells;' ' AO cells.' ''
D. Syncytium-forming viruses (primate, bovine, feline and avian)." 9' '
E. Slow virus
Visna virus;' ' 's9 Progressive pneumonia virus:
9' Maedi virus.9'

11. Intracellular Particles

A. Intracytoplasmic particles from tumor tissues
Human leukemic cells,6 ,' Human breast cancer cells;'' Human brain tumor cells;' ' *' Human Xerodema
pigmenlosum cells;' ' Human polycyfhemia Vera marrow cell line;' Mouse myeloma.' a
B. Type€ virus infected cells
Producer
NC-37 cells infected by SiSV-1' ' and by GaLV;' ' Mouse spleen infected by R-MuLV;' ' Chick embryo cells in-
fected by MC-29;' '' BALB/3T3 cells infected by MuSV.' '
Nonproducer
BALB/3T3 cells;' K-BALB/3T3 cells;' ' Rat cells transformed by RSV;' ' Chick embryo;' O ' Rhesus monkey
placenta.' ''
C. Intracisternal A-particles
Mouse neuroblastoma.' ' *' '

*This table does not include all of the known virus isolates. It includes only viruses or virus-like particles reported to
contain transcriptase activity.

December 1975 291

 presence of virus related components or particles proper name is the term DNA polymerase of the
both intracellularly and extracellularly from virus in question since it indicates the source and
human tissues, and for synthesizing DNA probes the basic characteristics of the enzyme without
from RNA templates. One immediate application describing the mechanism of enzyme action. How-
was to work out techniques for identifying this ever, this presents a serious problem when dealing
enzyme in cells, and to distinguish it from other with nomenclature of a DNA polymerase believed
(cellular) DNA polymerases. Gallo and colleagues to be viral but for which no virus has yet been
reported the first polymerase activity identified identified. For this reason we prefer the term
in any cell that could synthesize DNA from an reverse transcriptase for a DNA polymerase
RNA template.s This was with human leukemic believed to be an oncornavirus enzyme. However,
blood cells. Later, this enzyme was partially the term is not used loosely. Several criteria must
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purified: distinguished from the known cellular be met (see Section 1I.E). This has been the name
*’
DNA polymerases: r8 91 * and shown to exhibit used by most workers and agreed upon at a recent
the properties of the viral p o l y m e r a ~ e ’ (see
~~ meeting on eukaryotic DNA polymerases.’
Section 1I.E). This finding stimulated further In theory, the catalytic action of a DNA
search for viral specific components in human polymerase can be divided into four types
tumor cells and in other biological systems. depending on whether the template and primer are
These components include RNA-dependent DNA DNA or RNA. These are RNA-primed RNA-
polymerases, nucleic acids, and group-specific directed DNA synthesis, DNA-primed RNA-
antigens. At present, it is clear that the virus- directed DNA synthesis, RNA-primed DNA-
like DNA polymerase in virus-like intracellular directed DNA synthesis, and DNA-primed DNA-
particles of some human leukemic cells is bio- directed DNA synthesis. As shown in Table 2,
chemically and immunologically closely related both RNA-primed and RNA-directed reactions can
to some known primate type-C RNA tumor viruses be sensitive to pancreatic ribonuclease A. In order
and some murine t y p e 4 viruses6-’ J (see below to distinguish among these reactions, it is
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for detailed discussion). These findings suggest necessary to perform careful product analysis. This
some relationship between an RNA tumor virus is especially important in searching for “reverse
and the development of human leukemia. Parts of transcriptases” using endogenous undefined
this review will place emphasis on the nature of primer-templates. These reactions can be distin-
the intracellular reverse transcriptase and its guished by the density of the product in cesium
relationship to known viral reverse transcriptase sulphate gradients. If the size of primer molecules
and cellular DNA polymerases. is relatively small compared to the size of DNA
product, all RNA-directed reactions should have a
C. Nomenclature DNA-RNA hybrid density (1.55 g/ml) irrespective
Many names have been used in the literature for of the nature of the primer, while the density of
the viral DNA polymerase. This is due in part RNA-primed reaction products depends on the
to the fact that the enzyme is able to catalyze nature of the template. If the products are
DNA synthesis using either DNA or RNA as a denatured by heat treatment, all DNA-primed
template but also using an RNA primer (initiator). reaction products have a DNA density (1.45 g/ml)
For example, the term, RNA-directed DNA poly- whle RNA-primed reaction products have a
merase or RNA-instructed DNA polymerase is density slightly heavier than DNA. The deviation
used in order to emphasize the role and the nature of the density from DNA depends on the size and
of the templates since the word “dependent” does sequence of both RNA primer and DNA product.
not indicate that the RNA template is transcribed. The alkali treatment of all reaction products
Sometimes the term “RNase sensitive DNA poly- should release products only with the density of
merase’’ is loosely used to represent RNA- DNA. Another method to identify the RNA or
dependent DNA polymerase. The term reverse DNA primer is to determine the transfer of (u-~’P
transcriptase, which reflects some attachment to of deoxyribonucleoside triphosphate onto ribo-
the classical “central dogma” of molecular nucleotide or deoxyribonucleotides. If proper
biology, is the most conventional name. The primer-templates are used under proper condi-

292 CRC Critical Reviews in Biochemistry

 TABLE 2

Four Possible Types of DNA Synthesis Reactions with FWA or DNA as Template or Rimer

Product Analysis
~ ~~ ~~~

Name of Reaction Density in Cs,SO,Gradient**

Diagrammatic* RNase Not After After a-” P Deoxyribonucleotide

Name presentation sensitivity treated heat alkali phosphate transfer to

RNA-primed
RNAdirected
-
mvvvw4w
*---
Yes 1.55
>1.45
<1.55 1.45 Ribonucleotide
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DNA-primed
RNAdirected*** --- Yes 1.55 1.45 1.45 Deoxyribonucleotide
RNA-primed >1.45
DNAdirected ---- Yes 1.45 <1.55 1.45 R ibonucleotide
DNA-primed
DNAdirected No 1.45 1.45 1.45 Deoxyribonucleo tide

*m:RNA; -: template or primer DNA; --- newly synthesized DNA.

**DNA density: 1.45; DNA-RNA hybrid density: 1.55.
***Not identified in any natural system.

tions, viral DNA polymerase can catalyze all four from cell membranes, and is often able to cause
types of reactions. Some eukaryotic cellular DNA tumors in animals or transformation of cultured
polymerases are able to catalyze RNA-directed cells. Structurally, it consists of an outer
reactions when synthetic homopolymers are used membraneous envelope containing lipid and
’
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as primer-templates but not when natural RNA is glycoprotein’ and an inner core containing a
used (see below for detailed discussion), but the central dense nucleoid bounded by an inter-
transcription of natural RNA is not unique to the mediate membrane.’ ’
The size of this particle is
viral DNA polymerase since E. coli DNA poly- about 100 mp in diameter. Reverse transcriptase
merase I can also transcribe some natural is complexed with HMW RNA within the core
11 12
31
including viral HMW RNA if a high
‘
’
structure.’ I-’” Morphologically, ext racellular
concentration of the enzyme is used.’ ’’ oncornaviruses are classified into B-type, C-type,
With these biochemical and biological concepts and many other undefined types.’ The B-type
in mind, in this review the names “viral DNA particles have an envelope covered with tiny pro-
polymerase” and “reverse transcriptase” will be jections 5 to 10 mp long and an eccentrically
used on most occasions. Other nomenclature placed electron dense nucleoid, while the C-type
will be used when it is deemed necessary in the particles have a relatively smooth envelope (with
context of the discussion. smaller projections) and a central electron dense
nucleoid. This classification has been used to
name many newly found particles. However, these
11. EXTRACELLULAR REVERSE morphological criteria are rather subjective and
TRANSCRIPTASE often lead to many controversies in the absence
of a functional assay (Schidlovsky, personal
A. Extracellular Virus and “Virus-like” Particles communication). Due to the finding of a number
From a functional point of view, an animal of particles containing both reverse transcriptase
virus is defined as a membrane bound nucleic and HMW RNA in a variety of biological systems
acid-polypeptide complex which is able to repli- (see Table l), and due to limited availability
cate itself in a permissive host and produce of biological testing systems, tumorigenicity
progeny of its kind. This definition is based on the has not been a “sine qua non” of the RNA viruses
existence of extracellular, transmissible particles. containing reverse transcriptase. During this
In addition to this, an RNA tumor virus is avirus period, many names have been employed, such
which contains a HMW RNA,’ l 4 which buds as leukovirus, oncornavirus, rousvirus, retra-

December 1975 293

 virus, ribodeoxyvirus, RNAjDNA polymerase 1. Reaction Conditions
containing virus, or rnadnavirus.
Each new terminology has its emphasis and its a Pennention of the Virus Particle
bias. Here we will call them RNA tumor viruses Virus particles are generally made permeable by
oncomaviruses. Since the major reason for the treatment with low concentrations of nonionic
intense study of these viruses is the ability of at ’
detergents3,4 34 or ether.4 Requirement for this
least some to cause neoplasia. In fact, those “partial disruption”, in fact, has been used as an
capable of transforming in vitro are among the indication of the internal location of the viral
most efficient transforming agents known. Every polymerase. If the virus preparation is repeatedly
strain of polio virus does not cause polio yet frozen and thawed, treatment with nonionic
we call them polio virus. Moreover, “RNA tumor detergent may not be required. Ether extraction is
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virus” is the term in traditional use and is still infrequently used due to low yield of endogenous
the most commonly used nomenclature. For enzyme activity. This low yield of activity is not
these reasons, we maintain the “older” names due to an inactivation of the viral enzyme, rather
RNA tumor virus or oncornavirus. The term it is due to the destruction of the primer-template
“virus” will be reserved only for those structures (see below for discussion). Almost all nonionic
which have a replicating ability in a biological detergents are effective in partially disrupting virus
system. The term “particle” wiU be defined solely particles. Among them Nonidet P40,@ Triton
based on biochemical and physical criteria. In X-loo,@ and Sterox SL@ are most commonly
other words, the term “virus particle” does not used. Each nonionic detergent has a narrow range
imply any biological activity but it does not of optimal concentrations.“6 ,’’’-’ 2 4 High
exclude the possibility of a biological activity. concentrations of Triton inhibit the endogenous
With these definitions, the extracellular reaction. This is not due to dissociation of the
particles are classified into five categories (Table enzyme from the primer-template, since in a
1). They are the conventional RNA tumor viruses system using purified enzyme and viral 70s RNA,
(type-C and B), MPMV, syncytium-forming virus, DNA synthesis is unaffected by the presence of 2%
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and slow viruses of sheep. Little is known re- Triton X-100.’ 2 4 The more likely explanation for
garding the reverse transcriptase of syncytium- this inhibitory activity by nonionic detergent is
forming viruses and slow viruses of sheep. that it promotes the release of nuclease activity
which destroys t h e endogenous primer-
B. Reverse Transcriptase Activity in C d e Virus template.’ ’’,’’ s If an exogenous synthetic
Lysates primer-template is added to the reaction, DNA
Viral reverse transcriptase is located in the core
structure of viruses or virus-like particles’ ’ ,’’’’ synthesis continues even in the presence of rela-
tively high concentrations of Triton X-100.’24
and is complexed with viral nucleic acids.’ ’ This would then suggest that the enzyme is stable
In order to carry out DNA synthesis, it is in these concentrations of nonionic detergent. The
necessary to make the core permeable to the optimal detergent concentrations for endogenous
exogenous nucleotides and/or primer-template or reactions vary with viral purity, mode of storage,
to purify the enzymes from the virions. When age, and type of virus. Therefore, it is necessary to
DNA synthesis is directed by an endogenous RNA determine the optimal concentration for each
template, the system is generally called “endo- batch of virus. The concentration of Triton X-100
genous DNA synthesis reaction” or “endogenous used as reported in literatures ranges from 0.01%
reaction.” The reaction is at least partially sensi- to 0.2% and of Nonidet P40, from 0.1 to l%(see
tive to pancreatic ribonuclease A. In general, the a recent review by Green and Gerard’ ). These
endogenous reactions require partial disruption of ranges include both the reaction with and without
the particles, a divalent cation (magnesium or the addition of exogenous primer-template. In
manganese), and all four deoxyribonucleoside general, in a reaction with endogenous primer-
triphosphates, and is stimulated by reducing template, the optimal nonionic detergent
agents, salt, and phosphatase inhibitors. The concentration is below 0.05%.
enzyme in crude lysates also can use exogenously
added synthetic or natural primer-template for
DNA synthesis. A confirmation of RNA-directed b. Rimertemplate
DNA synthesis is dependent on a biochemical (1) Endogenous Prim=-templates
analysis of the reaction product.“2 Reverse transcriptase is defined by its ability to
294 CRC Critical Reviews in Biochemistry
 catalyze DNA synthesis utilizing viral RNA as catalyzes DNA synthesis by transcribing the
template. The basic observations are RNA strand of these duplex hybrid struc-
tures'41,142 and the DNA strand with a 3'-OH
1. The reaction is sensitive to RNase? j4 end serves as primer for the initiation of DNA
2. The newly synthesized DNA is associated synthesis.' Some synthetic double-stranded
with viral HMW RNA;' ' ' RNA such as (rA),,,*(rU),, and (rI)m-(rC) and
3. The purified DNA product can back some synthetic DNA such as (dC)m*(E)n,
hybridize to the HMW RNA: ' (dC), - (dI), and (dA=dT), are utilized but with
4. HMW RNA can be used as primer- poor Native DNA and de-
template by purified reverse transcriptase.' 2 4 ' natured DNA are also generally poor primer-
I 2 8-1 3 2 templates. Native DNA activated by a partial
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digestion with DNase I'46 is utilized but not as

The details of product analysis will be well as by most cellular DNA polymerases (both
elaborated further in the next section. The evi- eukaryotic and prokaryotic). (dT), (rA),
dence that RNA serves as a primer for reverse is utilized efficiently by some cellular DNA
transcriptase to catalyze DNA synthesis using polymerase and also by reverse transcriptase.
endogenous template is based on the observation The capacity to catalyze (dT), synthesis with
that DNA products are covalently linked to an crude cell extracts or with partially purified
6 primer is a species of 4s RNA,
~ ~ ~ . 1 3 3 - 1 3 e enzymes with this hybrid has been repeatedly and
which associates with HMW RNA by a hydrogen incorrectly used as a sole criterion for the presence
bond.' 3 7-1 3 9 of reverse transcriptase' 3' 9' '
(see below).
Endogenous reactions can also be stimulated by
the addition of DNA primer such as (dT)oligo and
(2) Exogenous Primer-template (dG)o,igo.14991'O The reaction stimulated by
The ftnding that some synthetic hybrids are (dT)oli led to the finding of (rA), sequence in
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extremely efficient primer-templates for reverse viral &A.' ' I-' 5 4

transcriptase greatly enhanced the sensitivity of While (dT)ollgo*(rA)n is the most efficient
the assay for reverse transcriptase. This is especial- p rime r - t e m p 1a t e f o r reverse transcriptase,
ly valuable for two purposes: in searching for (dT)oligo *(dA), is virtually completely inactive as
reverse transcriptase activity in a crude prepara- a primer-template. Therefore, the preference of a
tion and for estimating and detecting extracellular DNA polymerase to using (dT)oli o*(rA)n as
viruses. However, due to the lack of specificity of primer-template over (dT)oli ..(dAfn has been
the synthetic hybrids for the viral DNA poly- suggested to be one useful (%beit not absolute)
merase, application of this finding should be criterion for distinguishing reverse transcriptase
performed with c a ~ t i o n , ' ~and conclusions made from other DNA polymerase,' " but since
only after a number of criteria for reverse trans- (dT)oligo.(rA)n is not specific for reverse tran-
criptase have been satisfied (see Section 1I.E). As scriptase (normal cellular DNA polymerases P and
with other DNA polymerases, single-stranded y also utilize this hybrid as primer-template,'s6
homopolymers (DNA or RNA) are not used as especially in the presence of Mn"), care must be
templates by reverse transcriptase in the absence exercised in drawing conclusions and other criteria
of primers, but additionally, double-stranded must be satisfied. For example, the ratio of ability
synthetic DNA or RNA homopolymers are also of DNA polymerase y to utilize (dT)o,igo.(rA),
poorly utilized.' Hybridization of some over (dT)oligo*(dA)n is higher than 1O:l when
single-stranded homoribopolymers with Mn++is used as divalent cation. However, this ratio
complementary deoxyribopolymers or oligodeoxy- is not maintained when Mg" is used as divalent
ribonucleotides turns these synthetic duplex cation.' s 7 (dC)oli (rC), has been suggested
molecules into extremely efficient primer-templates as the only spechc synthetic primer-template
for reverse transcriptase. Examples of these for reverse transcriptase,' but this is clearly
-
are (dT),,, * (rA),, (Wok (rA),,, (dC)oligo
PrI),. The efficiency
not absolute with purified enzyme systems since
(rC),, and (dC)oli recently purified cellular DNA polymerase y
of these primer-tempfates varies with the type -
was shown to use (dC)oligo (rC), as primer-
of virus.' Reverse transcriptase asymetrically template.' '
December 1975 295
 c. Divalent ations reduction. The absolute requirement of four
Like other DNA polymerases, reverse tran- nucleoside triphosphates indicates that reverse
scriptase requires either Mg* or Mn" for catalytic transcriptase is able to transcribe heteropolymeric
activity!' ,48 In general, the mammalian (in- portions of viral RNA, and that its activity is not a
cluding. pri.mate) type-C RNA tumor viruses form of terminal addition. The concentration of
show greater activity with Mn", while the avian nucleoside triphosphate used in the endogenous
viruses, the typeB virus of mice, and Mason- reaction by different laboratories vanes greatly. It
Pfizer monkey virus show a preference for Mg". ranges from 0.05 mM to 8 mM for unlabeled dNTP
This preference is sometimes altered, especially and 0.06 phi to 0.1 mM for labeled dNTP (for a
when the purity of the virus is not well defined detailed summary, see Reference 126). No
and also when exogenous primer-templates are systematic study of the Km of each dNTP in an
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used. For example, Baltimore and Smoler endogenous reaction has been reported yet. The
reported that (dG)oli o-(rC)n is a better primer- effect of different amounts of the dNTP
template for R - M ~ L Qreverse transcriptase with (especially the ratio of unlabeled dNTP t o labeled
Mg" and that R-MuLV reverse transcriptase dNTP) on the kinetics of DNA synthesis also
prefers Mn++ over Mg" when (dT)oligo*(rA)n is remains to be studied. Recently, Rothenberg and
used as primer-template. In spite of these her colleagues observed that the high concentra-
preferences, the use of Mn" in endogenous tion of dNTP (as high as 5 mM) is essential in
reactions as the divalent cation yields more obtaining endogenous DNA product with higher
extensive transcription of the viral genome.' ' molecular weight (personal communication).
The optimal concentrations with Mn*, are narrow Nucleoside diphosphates and monophosphates are
(around 0.5 to 1 mM) while the Mg" optima are not substrates for the enzyme. Sometimes ATP
broad (around 5 to 10 mM). The optimum and CTP have been included in the reaction
concentration also depends on the type and purity mixture and reported to stimulate DNA synthesis,
of virus and on the presence of a chelating agent, although not incorporated into DNA.' 6 3 One
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e.g., EDTA. Due to the complexity of the endo- possible interpretation of this result is that AMP
genous reaction, it is not known 'whether the and CMP are incorporated into the CCA termini
presence of both Mg" and Mn" have a synergistic of 4s primer molecules'64 since a tRNA nucleo-
effect on the DNA polymerase activity. However, t i d y l t r a n s f e r a s e i s f o u n d b o t h in
when an enzyme prefers Mn", the presence of mammalian' 6 5 ,' and avian type-C RNA tumor
Mg" at a concentration higher than 1 mM is viruses.''
usually inhibitory. Other cations have also been RNA of RNA tumor viruses contains a track of
studied such as Cu", Hg", Co*, Zn", Cd", Ni", (rA&.' ''-''' The (rA), sequence is not usually
and Ag+.16' None of them are helpful for DNA transcribed in the endogenous reaction.' a j1

synthesis in the endogenous reaction, but zinc ion However, occasionally and for unknown reasons
has been reported to be associated with the native the (rA), sequence is extensively transcribed
polymerase.' I *9' (Reitz, M. S., unpublished observation). Hence,
it is advisable not to use dTTP as the radioactively
labeled dNTP for verifying transcription of the
d. Deoxyribonucleoside Triphosphates RNA heteropolymeric regions.
For a complete reaction, all four dexoy-
ribonucleoside triphosphates are required: dATP, e. Monodent Gztionsand Reducing Agents
dGTP, dCTP, and dTTP. Omission of one or more Either KC1 or NaCl stimulates DNA synthesis
deoxyribonucleoside triphosphate results in reduc- with an optimal concentration around 0.05 to 0.1
tion in DNA synthesis. The magnitude of the M.The optimal concentration has a broad distribu-
reduction depends on the purity of the virus and tion and varies depending on the buffer used in the
sometimes on nucleoside triphosphate. In general, virus suspension, the purity of the virus prepara-
omission of one deoxyribonucleotide results tion, and the type of virus. A salt concentration
in about a 70% reduction and omission of more higher than 0.2 M is generally inhibitory. There
Lhan two deoxyribonucleotides a 90 to 10W appears to be little difference between KCl and

2% CRC Critical Reviews in Biochemistry

 NaCl. The concentrations reported in the litera- template, the products of an endogenous reaction
tures range from 0 to 0.12 M.’ ’ should be carefully analyzed and meet the
The most commonly used reducing agents are following three criteria:
DTT and P-mercaptoethanol. Initially, DDT was
reported to be essential for DNA synthesis.“ a. The reaction products are DNA.
However, this is debatable when the purity of The fact that the reaction requires all four
the virus is not well defined. The concentrations deoxyribonucleoside triphosphates suggests that
reported range from 1 mM to 300 mM. the product is DNA. More definite proof is
obtained by showing that the product is semitive
f. pH and Ternperoture Optima to DNase, bands at a density of 1.45 g/ml in a
The reported values of pH optima range from cesium sulphate equilibrium gradient, and is resis-
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7.5 to 8.3. The temperature optima of the endo- tant to RNase and alkali. Generally, the size of the
genous reaction using endogenous primer-template product is small (about 4 to 6S), even though the
seems to reflect the temperature of the host template is large. Sometimes the product is so
cells. For mammalian virus the temperature small that it is not acid precipitable upon dissocia-
optima is about 37”C4,4’ and for avian virus tion from the primer-template.’ 7 2 The presence
’
40°C.3 3 s 9 4 However, when exogenous primer- of nuclease, predegradation of template, and non-
templates are used with crude enzyme prepara- ‘specific attachment of the radioactively labeled
tions, the optimal temperature vanes with the nucleotides on the filters are among the possible
thermal stability of the primer-template.’ 7 0 For interpretations of these findings. This ’ might
example, when (dT)o,igo.(rA), or (dT), .(rA), account for some loss of activity upon heat
is used, the optimal temperature is 25°C to 30°C. denaturation or alkaline treatment.
b. The DNA products are associated with
g. Ribonuclease Sensitivity primer-template RNA.
The original definition of reverse transcriptase A simple method to demonstrate that the DNA
For personal use only.

is based on its sensitivity to ribonuclease A in an product is associated with HMW RNA is (1) to
endogenous reaction3 34 since the template is band the nucleic acids extracted from a short-term
single-stranded RNA. The proper method to test reaction (no more than 5 min) in a cesium.
RNase sensitivity is to use a relatively low ribo- sulphate equilibrium density gradient and show
nuclease concentration (10 to 20 pg/ml) and that the labeled DNA moves as RNA (since the
relatively high salt concentration (0.1 to 0.2 M RNA is large and the DNA small); (2) to show that
KCl or NaCl). If the concentration of salt is too the DNA product cosediments with HMW RNA in
low and the nuclease concentration too high, a glycerol velocity gradient. This method was
degradation of RNA in a hybrid form may occur. named the “simultaneous detection” technique by
If this is the case, it will be difficult to determine Schlom and Spiegelman’ ’ (for simultaneous
whether the RNase sensitivity is due to degrada- detection of “viral” RNA and reverse tran-
tion of a requisite primer RNA in hybrid form or scriptase) and is frequently used as an initial
due to degradation of a single-stranded RNA indication of the existence of virus-related parti-
template. As shown in Table 2, this is especially cles, especially those isolated from cellular cyto-
important when one deals with RNA-primed DNA- plasm (see below for more detailed discussion).
directed DNA synthesis reaction. An example of Although HMW RNA is unique to viral RNA, there
such reaction is the cytoplasmic particulate frac- are many other reasons that could explain the
tion isolated from peripheral blood lymphocytes presence of radioactivity in the HMW region. One
stimulated by phytohemagglutinin (PHA).’ 7 ’ 7 2 should remember that a positive result from this
In this case, sensitivity of an endogenous reaction method may be used as an indication but not as a
to RNase could lead to an erroneous conclusion conclusion conclusion for the existence of a
that it was RNAdirected. complex of the viral polymerase and nucleic acids.
A firmer conclusion can be obtained if one isolates
2. Analysis of Endogenous Reaction Products DNA from the HMW region and hybridizes this
Since the most important characteristic of DNA back to the purified HMW RNA or by more
reverse transcriptase is its ability to catalyze the thorough product analysis (see below).
synthesis of DNA directed by natural RNA Another method to show that DNA products

December 1915 297

 are associated with RNA is to measure the density to use labelled dNTP's other than dTTP. It is still
of the DNA product before and after dissociation possible that the DNA product is linked to (dT),.
from the primer-template. This can be measured in Molecular hybridization is used to show that the
a cesium sulphate equilibrium gradient in which sequences maintained in the DNA product and
DNA, RNA-DNA hybrids, and RNA band at 1.45, viral RNA are complementary. The quantity
1.55, and 1.65 g/ml, respectively!' 9'7 3 With this of hybrid can be determined by banding the
method, DNA products purified directly from the hybrid in a cesium sulphate density gradient4 7 3
9'

reaction mixture should band at the hybrid region in which the DNA bands in the RNA region,
if the size of the DNA and RNA are comparable or by resistance of the hybrid to S1 nuclease,'"
at the RNA region if the size of the DNA is and by retention of hybrid to a cellulose filter
relatively smaller than that of the RNA. If the to which the RNA is covalently bound.' ''
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products are denatured by heating or by treatment

with formaldehyde or with DMSO,the products
should then band at either the DNA or RNA 3. Some Properties of the DNA Product
region depending on the nature of primer and/or a. Extent of Transcription of 70s RNA
on the size of the RNA primer. If the products are Generally, the DNA products synthesized in
treated with alkali or RNase, they should band at uitro do not uniformly represent the viral 70s
the DNA density on a cesium sulphate gradient. RNA genome. Duesberg and Canaani reported that
At early time periods of DNA synthesis, the RSV RNA was completely copied in an endo-
products are associated with template RNA by genous reaction but 85% of the DNA product was
hydrogen bonds and with primer by a covalent complementary to only 15% of the viral
bond, 1 73-1 7 5 The DNA product naturally moves
genome."' The experiment was performed by
with RNA. However, when DNA synthesis is measuring the degree of protection of "P-RNA to
prolonged, in most cases the DNA products are RNase after hybridizing excess amounts of DNA
either released as single-stranded DNA or con- product to viral RNA. The ratio of amount of
For personal use only.

verted to double-stranded DNA.' 74-' This con- DNA to RNA by weight was more than 1OO:l.
version can be inhibited in the presence of a high Similar findings were reported by Bishop et al.
concentration of actinomycin D (50 to 100 with RSVla3 and Gelb et al. with mammalian
pg/ml).' 74-' Probably reverse transcriptase, viruses' using reassociation kinetics.' IsWith
ribonuclease H, and other specific nucleases are the protection experiment, the products from
required for this conversion. many mammalian type-(: viruses have been found
c. Reassociation of the DNA product to the to represent more uniform copies of the viral
non(rA), portion of the RNA template. genome than those of avian viruses. For example,
In order to obtain conclusive evidence that Benveniste et al. reported that 70% of the viral
RNA is the template of the DNA product, it is genome w e protected from S1 nuclease digestion
necessary to demonstrate that the DNA product is by hybridizing with only a threefold excess of
homologous to the heteropolymeric portion of the DNA product to viral RNA.la6 It has been further
RNA template. There are two reasons for this. claimed that in R-MuLV, the whole 70s RNA was
First is to rule out the possibility of nonspecific uniformly transcribed.' Two conditions have
association of the DNA product to the RNA been reported to favor the extent of copying the
template; second is to prove that the product is HMW RNA. One is Mn*+ ion,' ,'* and the
not (dT),. As discussed above, the RNA of these other is the presence of actinomycin D in the
viruses contain a track of (rA),.' '-' 5 4 The (rA) reaction.' Recently DNA products have been
sequence is located at 3'QH end of the RNA' 7 8 used extensively as probes to study the mechanism
and is not generally transcribed by reverse tran- of viral replication to clarify the origin and
scriptase from avian' 6 8 or mammalian viruses.' 6 9 evolution of these viruses and to search for viral
Under certain circumstances the (rA), sequence is derived nucleic acids in viral transformed non-
copied, for example, when the DNA synthesis is producing cells. It is important to obtain methods
carried out in the presence of (dT)o,igo. The which allow for synthesis of DNA products which
simplest method to show that the entire product is uniformly represent the viral genome or which at
not just (dT), and hence to indicate that hetero- least indicate the fraction of the genome repre-
polymeric regions are at least in part transcribed, is sented.

298 CRC Critical Reviews in Biochemistry

 b. Size of the DNA Products ether, Flugel et al. found that there are two types
The size of the DNA products with the endo- of RNA-DNA linkages: a major bond, rU-dC and a
g e n o u s p r i m e r - t e m p l a t e is about 4 to minor bond, rA-dA.' 3 s The ether-disrupted B77
10s.' ,' 9'" 3' This is equivalent to only virus and R-MuLV also had two types of linkages,
about 100 to 150 nucleotides. Prolonged incuba- rCdC, the major bond and again rA-dA, the minor
tion does not increase the size of the DNA bond.' 9 3 To further elucidate the effect of
product, but most of the DNA is converted to disruption of the virions on the RNA-DNA
double-stranded DNA of similar size.' '' 74-' 7 8 linkage, Flugel et al. showed that when B77 virus
Sometimes the products are so small that, al- was treated with both NF40 and ether, only the
though they are acid precipitable when associated rA-dA linkage was found.'35 Another factor
which affects the RNA-DNA linkage is the age of
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with primer-template, they become acid-soluble

upon dissociation from primer-template.' 7 2 The the virus. Flugel et al. observed that in the
addition of a phosphatase inhibitor such as NaF to endogenous reaction of R-MuLV obtained after
the reaction mixture may prevent the synthesis of only 5 min pulse with labeled RNA precursor,
these small products. Recently, Guntaka et al. there were mainly rC-dC linkages with ether
reported that the final form of the DNA product disrupted virus. However, with virus harvested
of RSV upon infection of duck cells is a double- after 24 hr and disrupted by ether, tbere were
stranded helical circle with an estimated molecular both rC-dC and rA-dA linkages.
weight of six million,' and similar findings were
made by Gianni et al. in mouse cells infected by Clarification of these discrepancies and a final
murine leukemia virus.'" Therefore, there is a interpretation of the findings will depend on a
clear discrepancy between the in vitro endogenous detailed understanding of the sequence of the
reaction and intracellular proviral DNA synthesis. primer structure, and this has recently been
Lack of cellular factors or an inappropriate micro- achieved by Dahlberg and his associates. They have
environment might be reasons for the endogenous obtained the complete sequence of the primer 4s
For personal use only.

in vitro reaction. Apparently, high concentrations RNA (a species of tryptophan tRNA) from Rous
of dNTP (as high as 5mM)437 and conditions sarcoma virus. The sequence of 3'OH end was
unfavorable for nuclease activity, such as optimal shown to be -UCACCAOH.'~* These results, of
nonionic detergent concentration, tend to enhance course, support the rA-dA linkage. It is still
the rate of DNA synthesis and result in larger possible that other primers are used in the
sized DNA products. In some cases some products presence of some detergents. It will be important
with a molecular weight of 2.5 to 3 X lo6 daltons to determine if the primer used in vivo is the same
were obtained; however, these high molecular as the tryptophan tRNA primer discovered in
weight products are only a small portion of the vitro.
total product. When exogenous synthetic hybrids
are used as primer-templates, the size of the d. Base Composition of the DNA pt.oducts
product is larger than the RNA strand of the Several lines of information indicate that DNA
hybrid, but when activated natural DNA is used, products of the endogenous reaction are hetero-
the size of product is equivalent to the size of the polymers:
gaps digested by DNase.' 9 2
1. The DNA products are not (dT),;' *
c. Covalent Linkage of DNA Prbduct to RNA 2. The DNA products are complementary to
Primer heteropolymeric portions of viral HMW RNA both
Among 16 possible linkages between the DNA in avian4 ' and mammalian4' 9' '' virus
product and the RNA primer, Verma et al. found systems;
that only the rA-dA linkage was present in the 3. Nearest-neighbor analysis of the DNA
DNA product of the endogenous reaction from ''
product synthesized with one P-labeled deoxy-
AMV disrupted by NP40.134 The same finding ribonucleoside triphosphate and three unlabeled
was obtained from RSV,19' B77 virus,'93 and triphosphates showed that the DNA products are
R-MuLV' '4 when the virions were disrupted by h e t e r o p o l y m e r s c o m p o s e d o f all four
NP40. However, when AMV was disrupted with nucleotide~.~' 3'

December 1975 299

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For personal use only.

TABLE 3

Summary of Procedures for Purifying Viral Reverse Transcriptwe from Extracellular Partides

Fractionation procedures

Molecular
Solubilization Procedures** and Molarity weightf Presence of
Reference virus method* of Enzyme Eluation Buffer*** PH (X daltons) Purification RNase H

I. Conventional column procedures

~

Kacian et AMV 7% NP40 DEAECell(0.3G) KP (10) 7.2

al.' 0.8 M KCT
0.7% Na deoxy- CM-Seph.@C-50 KP (10) 8.0 40
cholate Pc (0.2G) KP (10) 8.0
GG (0.2) KP (10) 8.0 110 (V.S.) 6s
(95%) Seph. G200 (0.2) KP (10) 8.0 110 (SDS)
69 (SDS)
HA (0.2%) KP (10) 7.2
DNA Cell (0.2%) KP (10) 8.0
Baltimore and AMV 1% NP40 A-25 DEAESeph. Tris (25) 7.9 +
Smoler' '' (0.1G)
PC (0.3G) Tris (25) 1.9 NR 50 +
Gc (0.2) Tris 7.5 +
CG (0.5 Tris 7.5 +
Grandgenet t AMV 2% NP40 DEAECell (0.09G) KP (10) 8.0 +
et al.' '' (99%)
Pc PI (0.11G) KP (10) 8.0 +
PI1 (0.22G) NR +
GG PI (0.35) Tris 8.0 90 (V.S.) +
PI1 (0.35) Tris 8.0 160 (V.S.) +
Q 65 (SDS) NR +
B 105 (SDS) NR

*Number in parenthesis represents the percentage of enzyme recovered in supernatant after s o l u b h t i o n .

**w:Phosphodhlose; GG: glycerol gradient; HA: hydroxylapatite; Seph: Sephadex; ASP: amonium sulfate precipitation; PA: Peak A; PB: Peak B; PI: Peak I; PII: Peak 11;
I@: immunoglobulin G. The information in parenthesis represents the molarity of salt at which enzymes are purified in a velocity sedimentation and gel fdtration procedure or
the molarity of salt at which enzymes are eluted from ionic exchange chromatography. Some abbreviations are as follows: G: eluted by salt gradient; B: eluted stepwise by a
salt buffer, and others in case of velocity sedimentation and gel tiltration.
***KP: Potassium phosphate buffer; NaP: sodium phosphate buffer. The number in parenthesis represents the percentage of glycerol in the buffer.
tV.S.: Molecular weight estimated by velocity sedimentation in glycerol gradient or in sucrose gradient; SDS: molecular weight determined by a SDS polyacrylamide gel disc
electrophoresis; GF: molecular weight estimated by gel filtration.
t t N R : Not reported.
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For personal use only.

TABLE 3 (continued)

Summary of Procedures for Purifying Viral Reverse Transcriptase from Extracellular Particles

Fractionation procedures

Molecular
Solubilization Procedures** and Molarity weightt Presence of
Reference virus method* of Enzyme Eluation Buffer*** PH (X daltons) Purification RNase H

Malling et AMV 0.5% NP40 Sucrose gradient NR NR NR NR +

al.' ' DEAESeph. (0.075G) Tris (5) 7.5 +
Waters and AMV 0.8% NP40 GG (0.5) Tris 8.0 GS (V.S.)
0.5 M KC1 HA (0.04G) KP (15) 7.2
Frozen and thaw PI (0.0%) Tris (15) 8.0 50-1 10
PI1 (0.25G) +
R-MuLV GG (0.5) Tris 8.0 4 s (V.S.)
HA (0.025G) KP (15) 7.2
PC (0.25G) Tris (15) 8.0 100- 150 +
Hunvitz and AMV 0.4% Triton@ ASP (40%) Tris HCl 8.0
Leis' '' x-100
0.1 M NaCl PC PI (0.OSC) Tris (10) 8.4
(90-95%) PI1 (0.15G)
GG PI1 (0.05G) Tris 7.9 160 (V.S.) 80 NR
R-MuLV 0.4 Triton ASP (40%) Tris 8.0
x-100
0.1 M KCl Pc (0.22G) Tris (5) 8.0
GG (0.05) Tris 7.9 90 (V.S.) 75 < > 75 NR
Duesberg et PR- 0.5% Triton DEAE Cell (0.15- Tris (5) 7.4 170 (SDS) 8s 40
al.' a
' RSVIC x-1000.02 0.2G)
M KCl
SR- (75-80%) RNase treatment 110 (SDS) 6s NR
RSV/A
Faras et SR-RSV 1% NP40 (75%) DEAECell(0.4B) Tris (30) 7.2 (30%)
aL' ' PC PA (0.2G) NaP 6.8
PB (0.4G)
Seph. GlOO PA (0.4) NaP (30) 6.8 96 (GF) NR
PB (0.4) NaP (30) 6.8 105 (GF)
GG (20-40%) PA (0.4) NaP 6.8 5.3s (V.S.) 940
PB (0.4) NaP 6.8 6.2s (V.S.)
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TABLE 3 (continued)

Summary of Procedures for Purifying Virpl Reverse Transcriptase from Extracellular Particles

Fractionation procedures

Molecular
Solubilization Procedures** h d Molarity weightt Presence of
Reference virus method* of Enzyme Eluation Buffer*** PH (X daltons) Purification RNase H

Wang and PR- 0.5% Triton AS (45%)

Duesberg' 'I0 RSV-B x-100
PR- Seph. (3200 (0.4) NaP (30) 6.2 160 (V.S.) RSV NR
RSVC
K-MuSV .70 (V.S.) f
(KMuLV) [MuLV(MuSV)]
M-MuLV GG (0.1) Tris 8.1
MMuSV
(M-MuLV)
R-MuLV
Ross et. R-MuLV 1%Triton PEG-Dextran
a1.4 = 1 0.5
M KCl Seph. GlOO (0.3) Tris (20) 7.8 70 (GF)
1M Urea Pc (0.4G) Imidazob 6.5 100 NR
(20)
20% Glycerol GG (0.15) Tris 7.8 70 (V.S.)
Wu et al.' ' R-MuLV 1%Triton pc ( 0 . K ) Tris (20) 7.9 (20) +
K-MuSV 0.8 M KCl DEAECell(0.2G) Tris (20) 7.9 (20) 160 +
(K-MuLV) 20% Glycerol GG (0.3) Tris 1.9 70 (V.S.) +
Wu et a1.20 1%Triton DEAECell PI (0.05C) Tris (20) 7.9 (20) +
0.8 M KCl PI1 (0.14G) +
20% Glycerol Pc PI (0.2G) Tris (20) 7.9 (20) +
PI1 (0.2G) +
GG PI (0) Tris 7.9 70 (V.S.) +
(0.5) 7 0 (V.S.) +
PI ( 0 ) Tris 7.9 150 (V.S.) +
(0.5) 70 (V.S.) -+
Grandgenett RMuLV 0.2% Brij. 58@ Seph. GZOO (0.5) Bicine-N aOH 7.5
0.1 M NaCl DEAESeph. A-50 (0.2G) KP(10) 7.5 , 150
et a1.' '
Sephgros? 6B (0.1) KP (10) 7.5 480 (G.F.) NR
(0.5) 70 (G.F.)
135 (G.F.)
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TABLE 3 (continued)

Summary of Procedures for Purifying Viral Reverse Transcriptase from,ExtraceUulPr Partides

Fractionation procedures

Molecular
Solubilization Procedures** and Molarity weightt Presence of
Reference virus method* of Enzyme Eluation Buffer** * PH (X daltons) Purification RNase H

Weimann et F-MuLV Triton X-100 ASP (20-50%)

al.' Sonication DEAECell (0.15G) Tris (20) 8.0 +
FC
' (0.3G) Tris (20) 8.0 +
GG (0.3) Tris 8.0 123 (V.S.)
50-67 (SDS) +
Mulling et F-MuLV 7% NP40 DEAECell(0.3B) KP (20) 7.2 +
d I 0 1 +
0.8 M KC1 CM Seph.40 (0.3B) KP ( 20) 8.0
GG (0.3) KP 8 .o 84 (V.S.) (SDS)
45 -56 +
DEAECell(0.07G) KP (20) 7.2
PC (0.2G) KP (20) 1.2
Howk et al." RIII 2% Triton PC A (0.18G) Imidazole 6.8 (20)
Milk x-100 (20)
B (0.3G)
Seph. GlOO (0.3) Tris (20) 7.5 NR NR NR
NIH 2% Triton FC
' (0.3G) Imidazole 6.8
swiss x-100 (20)
BALB Seph. G150 (0.3)
Milk
MPVM
GaLV
Abrell and M-PMV 0.25% Triton +
Gallo' ' x-100
0.5 M KCI +
ssv-1 0.25% Triton DEAE€ell(O.OSG) Tris (5) 7.9 +
x-100 Sucrose Gradient (0.35) 110 (V.S.) 880 +
R-MuLV 0.5 M KCI PC (0.15G) Tris (20) 7.9 (20) 70 (V.S.) +
Viper Detergent PC (0.2G) KP (20)
c. type€ GG (0.5) Tris 7.8 109 (V.S.) NR
s
cn
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TABLE 3 (continued)

Summary of Procedures for Purifying Viral Reverse Transcriptase from Extracellular Particles

Fractionation procedures

Molecular
Solublization Procedures** and Molarity weightt Presence of
Reference virus method* of Enzyme Eluation Buffer ** * PH (X lo-' daltons) Purification RNase H

11. Affinity chromatographical procedures

Livingston SR-RSV 1%Triton Seph. GlOO (0.3) Tris (20) 7.8

et a P o 3 G-FeSV 1 M Urea IgG Sepharose Tris 7.8
(0.2M NH, OH)
R-MuLV 0.5 M KCl 0.3 M KCI 1%BSA
25% Glycerol (pH 10.6)
Livingston RAV-1 Seph. GlOO (0.3) Tris (20) 7.8
et SR-RSV
AMV IgG Sepharose (0.05M Tris 7.8
NH,OH, 1% BSA. 25%
Glycerol [pH 10.2-
10.3))
Gerwin and R-MuLV 1%Triton oligo(dT)Cell KP (20) 7.1
Milstein*' a (0.25G)
RD114 0.5 M KCI Pc (0.3G) Imidazole 6.5
virus (20)
Seph. GlOO (0.1) Tris (20) 7.8 50 (C.F.)
Marcus et AMV 1%NP40 Poly(rC)-AgUOse KP (10) 8.0 68 (SDS) 40 NR
aLIss (0.2G)
0.4 M Na deoxy- 105 (SDS)
cholate
0.4 M KCl
20% Glycerol GG (0.4) KP 8.0 6.6s (V.S.)
Chirikjian AMV
et al.'39 MMTV NR Pyran-Sepharose KP (20) 7.2 NR NR NR
(0.38G);(0.22G)
R-MuLV (0.2%) '
RSV (0.46)
FeLV (0.3G)
 C.Purified Reverse Transcriptase Activity from unsolubilized and high molecular weight
I. Purification components (such as nucleic acids) is ultracentri-
The purification of reverse transcriptase from fugation at about 50,000 xg (varying from 16,000
extracellular virus particles is relatively simple to 100,000 xg) for 30 to 60 min. With this pro-
compared to that of the purification of an enzyme cedure, about 7 5 to 99%of the enzyme is recovered
from whole cells, since there are few species of in the supernatant (see Table 3).
protein and nucleic acids in the virions, and no
other polymerase. The best purifications reported 6. Fractionation
are about 1,000-fold. These are reverse tran- (1) Conventional Fractionational Procedures
scriptase of Rous sarcoma virus''3 and of Ammonium sulphate precipitation is rarely
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MPMV.'96 However, due to a difficulty in used for fractionation of reverse transcriptase, but
obtaining large quantities of some purified viruses, it is used occasionally for concentrating the
not many viral reverse transcriptases have been enzyme. The commonly used procedures for
studied critically from a biochemical point of fractionation involve ionic exchange chromat.0-
view. For example, in most cases there is not graphy, gel filtration, and velocity sedimentation.
enough protein to determine its concentration and The combination of some of these procedures has
therefore the purity or specific activity cannot been shown to be very efficient for enzyme
really be determined. Table 3 shows a summary of purification.
the procedures and results of the purification of
viral reverse transcriptase reported to date (for a (a) Ion Exchange Chromatography
recent review, see Reference 197). The purifica- Since reverse transcriptase is a DNA-binding
tion procedures can basically be divided into two protein and most of the other viral components
phases. One is solubilization of the enzyme and are not, processing disrupted viruses through a
the other is the fractionation of the enzyme phosphocellulose column is usually sufficient to
molecules from other components. purify reverse transcriptase about 50- t o 100-fold.
For personal use only.

This degree of purification is useful for many

a. Solubilization practical purposes. However, in the authors'
Reverse transcriptase of RNA tumor viruses can experience, often some enzyme remains in the
be solubilized from virions by a treatment with flow-through due to adherence to nucleic acids. To
nonionic detergents such as Nonidet 40 or Triton overcome this problem, nucleic acids should first
X-100. As shown in Table 3, the concentration of be removed by DEAE cellulose column chromato-
each nonionic detergent used varies with the type graphy or a similar simple step. This process
of virus, with other components in the solubili- removes most of the loose binding nucleic acids,
zation buffer, and with laboratory preferences. but some tight binding nucleic acids (such as
The salt concentration also varies greatly in primer) are still bound to the eluted enzyme.
different reports. High salt concentration favors Almost all of the DEAE eluted enzyme can then
dissociation of the enzyme from other viral com- be adsorbed onto phosphocellulose. The PEG-
''
ponents including nucleic acids.' To enhance the dextran procedure also has been used to remove
solubilization of the enzyme, 1 M w e a l z 3 or 0.7% nucleic acids!' '
DEAE sephadex, CM sephadex,
deoxycholate' " 9 8 is sometimes added to the
9' hydroxyapatite, and DNA cellulose are other types
solubilization buffer or the procedures such as of ion exchange columns that have been used for
freezing and thawing' and sonication4 ' polymerase purification. When small amounts
1 9 6 9 2 0 0 are used. There is no reported evidence of virus are used for enzyme purification, the
that these additional reagents and procedures are presence of low concentrations of nonionic
necessary. Without exception, glycerol (con- detergent and reducing agent enhance enzyme
centration ranging from 5 to 30%) and a sulf- recovery.' 9 6 The salt molarity used in elution
hydryl agent (1 mM to 10 mM) have been in- of enzyme from these columns varies with many
cluded in all solubilization buffers. Solubilization factors, particularly with pH and compo-
is usually done at 0" to 4°C for $4 to 1 hr, al- nents of the buffer and also with the concentra-
though some prefer to incubate at 37°C. The tion of nonionic detergents. Therefore, compari-
initial step to separate the solubilized enzyme son of values from different reports is not

December 1975 305

 meaningful. Most of the enzymes purified through E. coli DNA polymerase was also adsorbed to the
ion exchange chromatography still contain some column but eluted at a lower salt concentration
other viral components, such as group specific compared to reverse transcriptase. Recently, Carl
proteins (Gallo and Wu,unpublished obseivation) Saxinger at the, National Cancer Institute has
and RNase H (see Table 3). developed a variety of polynucleotide affinity
chromatography columns with a cellulose matrix.
(b) Gel FUtration and VeIociry Sedimentation None of them have a unique specificity to reverse
Factors other than the charge of the enzyme transcriptase but a combination of various types
are used in the fractionation of reverse tran- of polynucleotide cellulose columns is potentially
scriptase, for example, size and shape. Thus, gel a very useful method for separation of reverse
filtration with Sephadex@ and velocity sedimenta- transcriptase from other cellular enzymes (Saxinger,
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tion with glycerol or sucrose density gradients are personal communication).

frequently utilized. Both Sephadex G-100 and
(3-200 are used. For velocity sedimentation, a 10 (c) Pyran-Sepharose Chromatography
to 30% glycerol gradient is common. Besides being Chirikjian et al. reported that pyranSepharose
a step in purification, this procedure can be used is an effective affinity column for the purification
to obtain a sedimentation size estimate of the of reverse transcriptase from several RNA tumor
enzyme. Since reverse transcriptase e n b m e viruses? 3 9 Analysis of iodinated proteins by SDS
aggregates in low salt," s201 it is advisable to gel electrophoresis revealed that the reverse tran-
purify the enzyme through these steps in a high scriptase from AMV could be purified nearly to
salt buffer for estimates of enzyme size. homogeneity by a single passage through the
In view of the ease of purification of reverse column. Since pyran-Sepharose is totally resistant
transcriptase from virus by conventional purifica- to nuclease digestion and is also stable at 4°C for
tion procedures, affinity chromatography is not several months, this could be a useful procedure to
particularly useful. However, a development of isolate reverse transcriptase from crude cell lysates.
In general, viral reverse transcriptases have a higher
For personal use only.

highly specific affinity chromatography would be

extremely useful in purifying reverse transcriptase affinity t o pyran-Sepharose than prokaryotic DNA
from cells. polymirases. Since the affinity of eukaryotic
cellular DNA polymerase to this column was not
(2) Affinity Chromatography shown, the application of this procedure to crude
(a) (dT)o~~o-Cellulose
Chromatography cell lysate system remains to be evaluated.
The use of (dT)oligo-cellulose chromatography
was based on the observation that (dT)oligo (rA), (d) Immunoadmrbant Chromatography
is a very efficient primer-template for reverse In immunoadsorbant chromatography, mono-
transcriptase. Gerwin et al. reported that cellular specific IgC to specific reverse transcriptase is
DNA polymerases are not adsorbed by (dT)oli o- linked to Sepharose.@ This then can be used to
cellulose, and that this type of chromatograp%y purify reverse transcriptases both from virus and
was unique for viral reverse transcriptase.202 Since infected cells.203 9204 IgG affinity chromato-
(dT)oligo*(rA), is not absolutely specific for graphy has been developed both in avian204 and
reverse transcriptase, unique adsorption of reverse in mammalian systems? O 3 One problem with this
transcriptase was perplexing. Indeed, other investi- method is the difficulty of obtaining sufficient
gators have found that it is sensitive to nucleases specific IgG for an efficient performance.
and that some cellular DNA polymerases do
adsorb to this column (Reference 424 and M. 2. Reaction Conditions
Sarngadharan, unpublished observations). Purified enzymes, of course, require exogenous
primer-templates for their catalytic activity. The
(b) (rC),-Agarose Chromatognrphy optimal conditions for DNA synthesis vary with
Among all of the synthetic primer-templates, the primer-template us'ed and also with the type of
(dG)oligo (rC), is probably the most specific virus.
for reverse transcriptase. Marcus e t al. reported
that with (rC),-agarose affinity column, a 40-fold a. DNA synthesis Using Synthetic Primer-template
purification was achieved.' 9 8 The eluted enzyme Most of the synthetic primer-templates that
still contained RNase H activity and p27 protein. stimulate crude enzyme from disrupted virions can
306 CRC Critical Reviews in Biochemistry
 TABLE 4

Some Biochemical Properties of Viral Reverse Transcriptase

Type C

Properties Avian Mammalian Type B MPMV

Preferred divalent
cations*
Activated DNA Mg* >> Mn* Mg* > Mn* NR** NR
(d A-dT),
(dT), - (rA), Mg* = Mn* Mn* > Mg* NR Mg" > Mn"
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(rA), (rU), Mn* > Mg* Mn* > Mg* NR NR

(dT)oligo * (rA), Mg* >> Mn* Mn* > Mg* Mg" > Mn" Mg" > Mn"
(dC)oligo * (rC), Mg* >> Mn* Mg" = Mn* Mg" > Mn* Mg* > Mn*
70s RNA Mg" > Mn* Mn" = Mg* NR Mn* > Mg"
70s RNA + (dT)ohgo Mg** > Mn* Mg" = Mn* NR NR
Molecular weight Q 70,000 70,000 110,000 110,000
0110,000
Separation of RNase H Not separable Separable NR NR
*The optimal concenbation of Mg* is around 5-10 mM and that of Mn" is around 0.5-1 mM.
The comparison is made based on their activity at optimal concentrations. The relative
preference for divalent cations differs in many reports. These represent the most frequently
used conditions recognized by the authors.
**NR:Not reported.

be used as primer-templates for purified enzymes. of prokaryotic or eukaryotic origin were able to
For personal use only.

Among them (dT)o1jgo*(rA)n, (dT), *(rA),, use it. This may be the most specific primer-
(dG)ollq.o.(rC)n, (dA-dT), are the most effi- template for viral reverse transcriptase.
cient. 3' Purified reverse trans-
9'

criptase efficiently uses the hybrid (dT)oligo * The divalent cation required for an optimal
(rA), as primer-template, but not its DNA DNA synthesis vanes with the primer-template
counterpart (dT)oligo*(dA)n.' 2 4 ~ 2 0 0 ~ 2 0 sDNA used and also depends on the type of the virus (see
polymerase y9 9 $1 s 6 5 8 1 and p of mammalian
91 Table 4). With (dT)oligo *(rA),, reverse trans-
and chicken cells' '' and bacterial DNA poly-
3" '
criptases from avian type-(: viruses' t y p e B virus,
rnerase2Os are able to use (dT)oligo-(rA)n as and MPMVZo8 prefer Mg" as divalent cation.
primer-template, but they also use (dT)oligo E n z y m e s f r o m m o s t mammalian type-C
(dA), with fair to high efficiency. Therefore, the viruses' 9 9 ,'O 1 prefer Mn" over Mg". For
lack of utilization of (dT)oligo * (dA), by a viral (dG)o;$2 .(rC), enzymes from avian type-C
enzyme provides a criterion to distinguish between virus, type-B virus' and MP-MV' ,' ' '
cellular and purified viral DNA polymerases. Since prefer Mg" over Mn", while the enzymes from
there has been no definite demonstration that mammalian type-C virus can use Mg" and Mn"
cellular C?!A 2olymerases (except DNA polymerase w i t h a b o u t equal efficiency.'9'~20' For
-
7 ) can use (dG)oligo (rC), as primer-template, this (dT);(rA),, SR-RSV enzyme' " uses Mg" and
is probably a better synthetic primer-template for Mn" with similar efficiency, AMV and MP-MV
reverse transcriptase under optimal conditions. enzymes use only Mg"128~19' and those of
(dT), .(rA), is commonly employed to screen for mammalian type-C viruses' 9 6 p2 slightly prefer
reverse transcriptase and for DNA polymerase 7 Mn* over Mg". For (rA)n-(rLJ)m and (dG)oli,o-
due to its high efficiency as primer-template. (Crn),." Mn" is a better divalent cation for
Recently, Gerard et al. found that a hybrid b o t h avian and mammalian typeC virus
structure between oligodeoxyribguanoylate and enzymes4 1' 9' For DNA primer-templates
poly(2'-0-methylcytidylate) [(dG)oligo (Cm),] such as (dA-dT)n or activated DNA, Mg" is
is an efficient primer-template for reverse trans- generally a better divalent cation for all type-C
criptase from avian, murine, feline, and primate virus enzymes tested.'23'19' For the same
viruses.' None of the cellular DNA polymerases primer-template the optimal concentration of
December 1975 307
 divalent cation varies with the type of virus. In termed a "reconstruction reaction." The purified
general, the optimal concentration for Mg" ranges reverse transcriptase from avian viruses (such as
from 5 to 10 mM, while for Mn*, it is from 0.1 to AMV and RSV) is able to transcribe heteropoly-
1 mM. Another factor determining the preference meric portions of homologous and heterologous
and optimal concentration of the divalent cations viral HMW RNA in a reconstruction reaction.' 2 4 9

is the configuration of the enzyme. For example, 1 2 8 s 1 2 9 9 2 1 2 The fraction of the HMW RNA
when R-MuLV is processed through a DEAE genome copied differs with various systems but is
column, two forms of the enzyme, probably low in all, for example, Faras et al.' *' found it to
monomer and dimer, are eluted. The optimal be 3%. Avian viral enzymes use murine viral RNA
concentration of Mn" for the two forms are as efficiently as avian viral RNA. The reverse
different when (dT)oligo*(rA), is used (0.5 mM
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transcriptase from mammalian viruses can also

for the monomer and 1 mM for the dimer). Upon transcribe avian and mammalian viral HMW RNA
further purification, both forms of the enzyme '
with similar efficiency.' 2 4 9-1 3 2 In all cases, all
become monomer and both have the same optimal four deoxyribonucleotides are required for syn-
concentration of Mn*+,i.e., 0.5 mM.20 ' thesis. In a reconstruction system, DNA synthesis
As discussed previously, the optimal tempera- is greatly enhanced by the addition of an exo-
ture is dependent on the thermal stability of the genous oligomer s u c h a s (dT)o,igo or
primer-template and also the species of virus. The ' ,'
(dG)oligo. 2 4 The DNA products have been
pH optimum for the purified enzyme reaction is shown to link to the 3'-OH end of oligomer'29>
similar to that ,of the endogenous reaction. As for ' and probably consist mainly of transcripts of
monovalent cations, Faras et al. reported that the the (rA), or (rC), (unpublished observation)
purified enzyme from SR-RSV is strongly regions of the viral RNA. Heat denaturation of
inhibited by relatively low concentrations of KC1; HMW RNA greatly reduced the primer-template
NaC1, or NH4Cl,'23 but in many instances, an ''
activity,' ,' however, addition of oligomer to
initial stimulation of activity is found followed by denatured RNA can partially restore the primer-
For personal use only.

inhibition at higher concentrations.' 2 4 9 6 In

9' t e m p l a t e . ' 4 9 d 5 0 Apparently, these added
most cases, concentrations higher than 150 mM oligomers serve as new initiators for DNA syn-
are inhibitory. A determination of the optimal thesis.
concentration for each case is advisable.
Several points are worth emphasizing in dis-
The amount of DNA synthesis increases linearly
cussing transcription of HMW RNA by viral DNA
with the concentration of primer-template but
polymerases: (1) The concentration of the enzyme
then decreases at very high concentrations. This
should be sufficient. This is particularly important
inhibition of DN.4 synthesis by higher concentra-
when only a small quantity of virus is available and
tions of primer-template can be partially overcome
by the presence of nonionic detergent.' 2 4 the amount of enzyme is estimated only by its
Possibly, the nonionic detergent prevents the ability to use synthetic primer-templates; (2) The
primer-template from collape from the tertiary quality of HMW RNA should be carefully
structure. monitored. Probably one of the most critical tests
In the endogenous reaction, DNA-directed is its ability to dissociate to 35s RNA upon heat
DNA synthesis is observed in addition to RNA- treatment; (3) Optimal conditions for HMW RNA
directed DNA synthesis? ' ' '
9' These two transcription are also important. In general, the
activities can be separated by a high concentration divalent cation, Mg" is preferable for enzymes
of actinomycin D which suppresses DNAdirected isolated both from avian and mammalian type-C
DNA synthesis.' 7 6 '' The purified enzyme viruses,' 2 3 2',2 1 3 b ut the MP-MV polymerase
3'

'
$'

is also able to catalyze both reactions.' ' ,'

"' prefers Mn".' The optimum concentration of
' ,'
4s Therefore, reverse transcriptase has Mg*' is around 5 to 10 mM. Sometimes, addition of
apparently both catalytic functions or at least 1 mM Mn++to a reaction mixture containing 5
appears to have the capability of catalyzing both Mg'* further enhances DNA synthesis;' ''(4) The
reactions. products should be DNA and should be carefully
analyzed to ensure that heteropolymeric portions
b. DNA Synthesis Using Natural RNA of RNA and not the (rA), portion are
When the primer-template added to purified transcribed.' ,' '
reverse transcriptase is viral HMW RNA, it is Similar to the endogenous reaction, the pro-
308 CRC Critical Reviews in Biochemistry
 ducts of the reconstructed reaction are also small method the gradient sedimentation should be
(about 4 to 7S).’ 36 ,’’ ’ The products are bound performed in a high salt buffer to prevent the
to HMW RNA by hydrogen bonds and covalently enzyme molecules from aggregating; in high salt
linked to the 3’-OH end of the primer buffer, the enzymes are relatively stable and
’’
molecules.’ ” The RNA-DNA linkage is rA* sediment at about 4.5s for the type I enzyme,
dA. 1 3 4 , 1 5 0 The primer is 4s RNA’ ’ ,’’ which 6.5s for type 11, and 5.5s for type 111. In low salt
has tryptophap accepting ability’ and slight buffer, the enzymes tend to aggregate and are
methionine accepting ability.’ 383’9 5 This tRNA somewhat unstable as judged by a poor recovery
can be isolated from purified HMW RNA after following gradient analysis.’ 9’ ’’>’
heat treatment of the HMW (8OOC). 2. Gel filtration - As with the velocity
Avian viral reverse transcriptase can also use sedimentation method, the molecular forms of
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many other natural RNAs as primer-template reverse transcriptase are affected by the salt
albeit not as efficiently as the 70s oncornavirus concentration of the buffer. Low salt buffer tends
RNA, e.g., f 2 viral RNA, Qp viral RNA, bulk E. to form aggregates of the enzymes. For example,
coli tRNA,’ ’ ’
I’ poliovirus RNA?’ influenza Nakajima et al. reported that aggregates of the
virus RNA, tobacco mosaic virus RNA, and ribo- enzymes with a molecular weight of 480,000 were
somal RNA.’49 Some natural RNAs are poor obtained when highly purified R-MuLV enzymes
templates for reverse transcriptase, but become were processed through sepharose 6B at low salt
very good primer-templates upon addition of concentrations (0.05 M KCl) (without removing
oligomer. Examples include poliovirus RNA,’ nucleic acid).’ ’ The same enzyme was found to
rabbit globin mRNA,’ ’ ’
9’ human globin have two lower molecular weight.forms (135,000
mRNA? ’ and calf lens crytallin mRNA.’ ’ and 70,000 daltons, respectively) when processed
Globin mRNA transcripts approach 9 to 1 0 s in at high salt concentrations (0.5 M KCI). Another
size (the size of globin mRNA), and they are method of obtaining a monomer form is to
homologous to the mRNA. DNA synthesized in perform agarose gel filtration in 6M guanidine-
’
For personal use only.

vitro from mRNA has been a useful probe in hydrochloride.’ By this method, the molecular
studying the regulation of gene expression, weight of both R-MuLV and R-FeLV was found
particularly of globin mRNA. to be about 70,000. As well as the possibility of
finding aggregate forms, it is still possible that
3. Struc;ural Properties polymerase may associate with nonpolymerase
Purified reverse transcriptase can be classified proteins. This could, of course, affect the mole-
into three types based on molecular weight and cular weight estimates. Another problem occurring
subunit structure which we have arbitrarily both with the velocity sedimentation and gel
defined as types I, 11, and 111. Type I reverse filtration methods is the difficulty in obtaining
transcriptase is a single polypeptide with a accurate sedimentation values for the marker
molecular weight of about 70,000. Viruses con- molecules. In fact, the molecular weight descrip-
taining this type of reverse transcriptase are tion of the monomer of type I reverse tran-
mammalian type-C viruses (see Table 3 and scriptase varies in the literature from 50,000202 to
References 51, 218, 220, 221) and avian 90,000.’
REV.’ ” ,’ ” Type I1 reverse transcriptase has 3. Polyacrylamide disc gel electrophoresis in
two subunits with a molecular weight of about the presence of 0.1% SDS - This is the most
170,000; the (Y subunit has a molecular weight of accurate method for estimating size of single
70,000 and the 0 subunit has a molecular weight chains of polypeptides. By this method, it is
of about 110,000. All the avian leukosis-sarcoma generally agreed that the type I reverse tran-
viruses contain this type of reverse transcriptase. scriptase has a molecular weight ranging from
Type 111 reverse transcriptase has 1 subunit with a 70,000 to 84,000’ ’’ ’’
3’ , the a subunit of type I1
molecular weight of 110,000. The viruses con- enzyme 69,000 daltons, the subunit of type I1
taining this type of reverse transcriptase are the enzyme 110,000 daltons, and the type 111 enzyme
murine type-B virus, MP-MV, and the viper type-C 110,000 daltons.
virus (Table 3).
Three methods are frequently used: The configuration of the enzyme molecules
sometimes depends on the method of purification.
1 . Velocity sedimentation in either a For example, when disrupted R-MuLV was pro-
glycerol or sucrose density gradient - With this cessed through a DEAE cellulose column with a
December 1975 309
 shallow salt gradient, two peak activities were neutralizing activity against reverse transcriptases,
obtained. Peak 1 eluted at 0.05 M KCl and peak I1 purified IgC has been a valuable reagent in
at 0.14 M.Peak I has a molecular weight of 70,000 distinguishing various types of reverse transcriptase
both in high and low salt gradients. Peak I1 and reverse transcriptase from cellular DNA poly-
enzyme consists of dimer or trimer forms at low merase, particularly the reverse transcriptase-like
salt and a monomer form at high salt in the enzymes isolated from some human cells.’ ,9 9

presence of nonionic detergent. Peak I1 enzyme x 2 , z 3 4 Table 5 summarizes the studies on the
differs from peak I in some of its character- serological relationships among various sources of
istics.’” When both peak I and peak I1 enzymes viral reverse transcriptase. Antibodies to about 10
were further processed through phosphocellulose different viral reverse transcriptase have been
columns, both eluted at a salt concentration of 0.2 obtained in various laboratories. They are classi-
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M KC1.’” Another example of a configurational fied into six groups according to their antigenic
change reported in the literature with respect to relationship with their corresponding homologous
purification procedure is that of AMV. When the enzymes:
AMV polymerase was processed through a DEAE-
cellulose column, a single peak activity was 1. Antibodies to GaLV, SSV, MuLV, and
obtained. However, when this peak activity was MuSV reverse transcriptase? 3 2 3’
yz

f u r t h e r processed through phosphocellulose 2. Antibodies to FeLV and FeSV reverse

column, two peaks of activity were obtained.” transcriptase.’
Peak I contains only a subunit, while Peak I1 3. Antibodies to cat (RD114)2131236 and
contains both a and fl subunits. Both peaks baboon (M7)” endogenous viral reverse tran-
contain reverse transcriptase and RNase H scriptase.
activities. Apparently, a subunit is the simplest 4. A n t i b o d i e s to ALV reverse tran-
functional unit for reverse transcriptase. The scrjptase.2 3 2 92 3 7 3 8
9’

putative fl subunit has yet to be isolated, and its 5 . A n t i b o d i e s to REV reverse tran-
For personal use only.

function is not known. However, it was observed scriptase.2 ”

that the “subunit” can change the mode of 6. Antibodies to MP-MV reverse tran-
action of RNase H activity from random exoribo- scriptase.’ s’ 4 0
nuclease activity to processive exoribonuclease
activity.22* Another factor that may affect mole-
Antibodies against reverse transcriptases of mam-
cular weight is digestion by a protease. Recently,
mary tumor virus, syncytium-forming virus, and
MollingZ2’ demonstrated that the subunit can
slow virus of sheep have not been reported. It is
be converted in vitro to an a subunit plus a
likely that some of these will become a new group.
fragment with a molecular weight of 40,000
There are some variations in the reports regarding
daltons by trypsin treatment or by aging of the the antigenic relationships among different reverse
enzyme at -20°C. The fl to a ratio decreases from transcriptases. For example, antibodies to SSV
4.1 to 0.43 upon trypsin treatment.229 In fact, reverse transcriptase sometimes partially inhibit
the fingerprint patterns of trypsin-partially. R-MuLV reverse transcriptase6 but sometimes do
digested a and Olp subunits were found t o be nearly not.’ Similarly, antibodies to MuLV reverse tran-
identical.” y 2 I Similar observations of change of scriptase neutralize the FeLV reverse transcriptase
enzyme molecular weight (from 84,000to 70,000) with varying degrees of potency, and antibodies of
following protease treatment have also been made
RD114 reverse transcriptase sometimes inhibit
with Friend virus reverse transcriptase, a type I SSV-1 and R-MuLV reverse transcriptase activity,
reverse transcriptase (Molling, personal com- but again sometimes do not. Normally, this varia-
munication). tion does not occur in the same preparation of
antiserum. Two factors contribute to this varia-
4. Immunological Properties tion: (1) The host animals have different sensiti-
Antiserum against reverse transcriptase can be vity in recognizing the antigenic determinants of
obtained either from animals bearing tumors inoculated antigen^.'^ I v Z 4 Therefore, it is im-
induced by murine type-C viruses, or more fre- portant t o try to prepare antisera in several species
quently from animals immunized with purified before the final determination of their antigenic
reverse transcriptase.’ ” Due to its specific relationship. (2) Purity of enzymes - unpurified

310 CRC Critical Reviews in Biochemistry

 TABLE 5

Summary of Reports on immunological Relatedness of Different Reverse Transcriptase

Source of antibody

Source of reverse MuLV FeLV

transcriptase GaLV SiSV MuSV FeSV RD114 M-7 AMV RSV(RAV-0) TDSNV M-PMV

GaLV ++++ u++ ++ 0 +I+ 0 0

GBr 1-3 tn+ ++++ 0 0 0
ssv ++++ ++++ ++ 0 0-+++ 0 0
C-MuV +tt+ 0-+
MuLV 0-+ 0-++ ++++ ++ 0-+++ 0 0 0
+ +
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RDll4 0-+ 0 ++++ 0 0

M-7 0 0-+ 0 ++t ++++
FeLV 0-+ 0 0-+ +u+ 0-+ 0 0
RaLV +tt ++ 0
HaLV ++ ++ 0
PK (15) Virus ++ + + 0
AMV 0 0 0 0 ++++ +++ + 0
RSV(R 4V-0) 0 ++++ ++++
SR-RSV 01 B-RSV 0 ++++ ++++
RAV-1 01 RAV-2 +++
B77 ++++
csv 0 0 ++++
TDSNV ++ ++ ++++
REV-T ++ ++ ++++
MPMV 0 0 0 0 0 ++++
MMTV 0 0 0 0 0
Visna virus 0 0
Viper virus 0 0 0
For personal use only.

Foamy virus 0 0 0 0 0
H L 2 3 virus ++++ ++++ + o+ 0-+ + 0
Her, 12 virus ++++

Class of antibody I I1 111 IV V VI

*Degree of neutralization activity, ++++, ++++, strong; ++ = moderate; 0-+ = weak; 0 = no activity.

components may induce antibodies that non- viruses and cellular DNA polymerases?
specifically inhibit or stimulate reverse transcriptase
activity. To obtain significant results regarding 1. Reverse transcriptases are only mini-
antigenic interrelationships among various reverse mally or not detectably related to each of the
transcriptases, a well-defmed assay system. should cellular DNA polymerases.
be carefully monitored. The optimal conditions for 2. Reverse transcriptases from type-C
antigen-antibody interaction, particularly the pre- RNA tumor viruses are not detectably related to
incubation time,’ the type and amount,of non- reverse transcriptases from typeB RNA tumor
specific protein carrier?37 and the salt viruses.
concentration should be determined.2 2 2 Another 3. Reverse transcriptases from type-C
method to study the interrelationships of the mammalian viruses are not detectably related to
different reverse transcriptases is by a blocking reverse transcriptases from avian t y p e 4 viruses.
test which is basically a measurement of 4. Reverse transcriptases from type-C
competition between a given test enzyme and the viruses obtained from various species of lower
homologous enzyme.222, 2 4 0 If a monospecific mammals (e.g., cats, rats, mice) are related but
antibody is available, the radioimmune assay is distinguishable.
another useful method. 5. Reverse transcriptases from the two
At present several general conclusions can be distinct “families” of primate type-C viruses, i.e.,
reached regarding the interrelationships among the endogenous type-C virus of baboons vs. the
antibodies from various sources of RNA tumor horizontally “moving” type C virus from gibbon
December 1975 311
 ape leukemias and from woolly monkey sarcoma located inside the outer envelope. If the activity
are distinct. associated with the particulate fraction is not
6 . Reverse transcriptases from the gibbon affected by protease, requires detergent t o detect,
ape leukemia virus (GaLV) and from the woolly and bands at 1.25 g/ml in a sucrose density
monkey (simian) sarcoma virus (SSV) are very gradient, it very likely is located in the core. There
closely related. are two reasons to suspect that many or all of the
7 . Reverse transcriptases from the endo- enzyme activities associated with the outer
genous feline virus, RD114, is closely related to envelope are derived from cells: (1) All of the
the endogenous primate virus (from baboons). reverse transcriptase containing viruses bud from
8. Reverse transcriptases purified from the host cell membrane and these membrane
the fresh blood cells of some patients with acute components can easily be associated with the
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myelogenous leukemia (AML) are very closely budding virus; and (2) No “purified” virus is really
related to reverse transcriptase of SSV and GaLV. free from cell debris. It is generally accepted that
9. The reverse transcriptases from three the enzymatic activities associated with the core
virus isolates obtained from blood and bone have some significant function and those located
marrow of one patient with A M L * ’ , ~ ’ ~are a1 inside the outer envelope may also have some viral
closely related to reverse transcriptases of SSV and specific function. However, it is important to
GaLV while a second viral component is related emphasize that as long as our knowledge regarding
to BaEV components. the mechanism of viral replication and its genetic
10. Reverse transcriptases purified from regulation is incomplete, it is presumptuous to
some patients with leukemia so far have not been assess a functional role to any enzyme activity
found to be related to reverse transcriptase from which is not novel to the virus. In this section, the
any animal virus tested (Gallagher, R. and Gallo, discussion will be limited to one enzyme, RNase
R., unpublished results). H, which may very well be relevant to the reverse
transcription pathway and at least in some cases
For personal use only.

D. Enzymatic Activities Associated with Extra- may provide additional catalytic activity for the
cellular Particles reverse transcriptase protein.
1. Virus Specific or Adventitious
Other than reverse transcriptase, some other
enzymatic activities have been reported to be 2. Ribonucleuse H
associated with virus particle preparations. These There are at least two mechanisms t o explain
include DNA ligase activity;44 DNA exonu- the conversion of RNA-DNA hybrids t o double-
clease activity: 4 4 nucleoside diphosphokinase stranded DNA during the course of synthesis of
activity: ’ nucleoside triphosphate phospho- the proviral DNA. One is by peeling off the
single-stranded DNA from the RNA-DNA hybrid,
tran~ferase,~ ATPase? 6-2 and protein
kinase and phosphate acceptor protein^;^ ,2 ’ and the other is by an enzyme activity which is
capable of degrading the RNA strand of a
DNA endonuclease,I4’ ,I4’ RNase,122s’2s, 2 s 1
RNA methylase,2 ” y 2 s nucleotide k i n a ~ e ? ~ RNA*DNA hybrid. This type of specific nuclease
has been found in calf thymus cells and is termed
ribonucleoside triph~sphatase;~ phosphoprotein
p h o ~ p h a t a s e s, 4~ l a c t i c d e h y d r ~ g e n a s e ; ~ ~
’ ’
ribonuclease H.’ p 2 Molling and co-workers
found such activity associated with AMV
h e ~ o k i n a s e , ~Ah4V
~ ~ stimulatory protein; ”
This observation was soon confirmed
aminoacyl tRNA ~ y n t h e t a s e*, ~ ribonucleotide
~
terminal transferase activity,’ ’ and ribo-
by Baltimore and S m 0 1 e r ’ ~ ~in AMV and was
nuclease H.2 y2 **’ ’ ’ The location of these
extended to some other mammalian typeC viruses
by Grandgenett et a1.l s6
enzymes in the virion is defined based on the
sensitivity of the activity to protease and the
necessity of nonionic detergent treatment of the a Ribonuclease H porn Avian Type-C Viruses
virions for detection of the activity. If the activity In avian myeloblastosis virus, RNase H co-
is detectable without nonionic detergent treatment purifies with reverse transcriptase through two
and is sensitive to pronase, it is probably located ionic exchange chromatographies and glycerol
on the outer envelope. If detection of the activity gradient centrifugations.22 4 p2 l2 The reverse
requires nonionic detergent treatment but is not transcriptase activity is eluted in two peaks from
detectable in the core fraction, it is probably the phosphocellulose column containing (Y and a@
312 CRC Critical Reviews in Biochemistry
 subunits, respectively. Both peaks contain RNase b. Ribonuclease H from Mammalian Type-C
H activity.227 The RNase H activity is inhibited Viruses
by antibody to AMV reverse transcriptase.’ ’ ,’
6’ Reverse transcriptase from mammalian viruses
These findings strongly suggest that RNase H and also contains RNase H activity, but the relative
reverse transcriptase activities reside on the same amount of RNase H activity t o reverse tran-
molecule. This is further supported by the fact scriptase activity is less than that in avian
that both activities were found in the core of the viruses.4 8 , 1 3 1,’ 5 6 , 2 5 7 , 2 6 9 The amount of RNase
virions.’’ A firmer conclusion of this relationship H also vanes with the type and preparation of the
was obtained by demonstrating that RNase H acti- virus. For example, the RNase H activity reported
vity is present in QI and 09 subunits isolated in a for R-MuLV is only one fourth to one eighth that
nondissociating disc gel electrophoresis,’ ’ and of AMV.’ RNase H activity of F-MuLV is three
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that RNase H activity isolated from two to five times less than that of avian viruses (F‘R
temperature-sensitive mutants of reverse trans- RSV-B, PR RSV-C), but two to three times more
criptase of RSV, LA335, LA337 is also than that of Ki-MuSV ( M u L V ) ! ~ ” ~ ~ RN ase H
temperature-sensitive.’ J 6 4 However, the activity in M-MuLV, however, has been reported
functional sites of RNase H activity and reverse to be equivalent to that of AMV.I3’ Since, in
transcriptase activity appear to be separable for general, the activity is relatively low, it is not
the following reasons: (1) RNase H activity both possible to detect activity if substrates of low
in endogenous reactions and with purified specific activity are used for the assay.’’ ’ ,’ 79

enzymes is inhibited more than 80% by 30 mM ’ 7 0 Similar to avian virus enzyme, RNase H acti-
NaF and 150 mM KCI, while there is little effect vity isolated from F-MuLV’ ’ and M-MuLV’ 3 2
on reverse transcriptase (2) Con- was found to be random exoribonuclease. How-
versely, reverse transcriptase activity is stimulated ever, Molling found that RNase H activity ob-
by nonionic detergents but these have no effect on tained from freshly purified F-MuLV enzyme
RNase H activity (our unpublished data); (3) (with a molecular weight of 84,000) contained
For personal use only.

Reverse transcriptase is much more heat labile processive exoribonucleolytic activity and this
than RNase 1-1.’ I’ activity became random exoribonucleolytic when
The avian viral RNase H activity is exonucleo- the molecular weight of the enzyme became
lytic and the digested products are oligomers, 70,000 upon storage or partial trypsin digestion
predominantly dimers.260 This is in contrast to (personal communication). In contrast to the
the cellular enzymes from E. coli,266calf thymus avian viruses, we found that RNase H activity is
cells, 2 6 o , 2 6 chicken embryo cells,’6 $’ ’ not present in the core of murine typeC viruses
human KB cells,26 and human leukocytes,26a when the core structures are prepared with non-
which are endonucleolytic and from which the ionic detergents and isolated in a sucrose density
products are predominantly tetramers. These two gradient.’57 We think that the lack of RNase H
findings have, therefore, been used as two criteria activity in the core probably is not due to a non-
for distinguishing cellular and viral RNase H. specific inactivation or inhibition of RNase H
However, in our judgment these criteria are not activity during the experimental procedure since
definitive because of the complexity of the RNase H activity was detected in the core of AMV
enzyme kinetics in vitro. A more definitive answer prepared by the same procedure. Furthermore,
may be obtained with antibodies to viral RNase H. enzyme purified from the core of R-MuLV did not
The role of the CC subunit of viral reverse contain any detectable RNase H activity. We
transcriptase is not known; it might have some believe, therefore, that RNase H activity and
regulatory role in the mechanism of action of reverse transcriptase do not reside on the same
RNase H activity. For example, Grandgenett et polypeptide molecule and not even in the same
a1.’28 reported that CY subunit has a random compartment of the virus. T w o observations
exoribonuclease activity which results in the further support the conclusion that the RNase H is
release of the substrate molecule after each chain separable from reverse transcriptase in murine
scission while the Orp molecule has processive viruses: (1) The ratio of RNase H t o reverse
e x o r i b o nucl ease activity which completely transcriptase tends to decrease during the purifica-
degrades one polyribonucleotide chain prior to tion procedures and varies with the batch of virus
initiating hydrolysis of a second chain. and the procedure of purification.’ Olafsson et

December 1975 313

 al. were able to dissociate further RNase H activity divalent cation. Both Mn" and Mg" are suitable
from reverse transcriptase activity in a sucrose for enzyme catalytic action, but they have
density gradient in the presence of high salt.'6 It different optimal concentrations. The range
is worth noting that some RNase H is easier to reported for Mn+*is 0.3 to 0.8 d. Mg" optimum
separate from reverse transcriptase from murine is about 5 mM.48 The molecular weight of
type-C virus than it is from avian viruses, and at mammalian viral RNase H is not known, although
least two RNase H activities were found in there are indications that it is smaller than but very
R-MuLV and K-MuSV(Ki-MuLV),' and close to the size of reverse trans~riptase.'~~
M-MuSV(MULV).'" In addition to these observa- Cellular RNase H is, in fact, about 70,000 t o
tions, the larger form of R-MuLV reverse tran- 80,000 daltons.' J ' The discovery of RNase H
scriptase contains much less RNase H activity than has prompted some speculations regarding the
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the LMW enzyme;"' (2) Antibodies to mam- mechanism of double-stranded proviral DNA
malian viral reverse transcriptases do not inhibit synthesis. The models proposed by Molling et
RNase H associated with purified reverse tran- '
al.,? Keller and Crouch,z6 and Leis et al.' 7 6
scriptase, although the purified enzyme used as all require participation of a host endonucleolytic
antigen contained both activities (Wu and Gallo, RNase H as well as the viral nuclease. They do not
unpublished data). This indicates that reverse explain the conversion of the double-stranded
transcriptase and RNase H have different antigenic RNA primer region to double-stranded DNA.
determinants.
E.Criteria for Viral Reverse Transcriptase
c. Miscellaneous Properties of RNase H In view of the frequent use of inadequate
Virus particles contain many types of nucleases, criteria to define intracellular reverse transcriptase
and it i s necessary to distinguish "pseudo" RNase and sometimes extracellular particle enzymes, it is
H from true RNase H. Normally, RNase H is important to examine carefully the criteria which
measured by the release of acid nonprecipitable are useful and/or specific for the definition of
For personal use only.

radioactive oligoribonucleotides from RNA-DNA reverse transcriptase. We use two approaches:

hybrids in which the RNA moiety is radioactively one is biochemical, and the other is immunological.
labeled. Three types of nuclease reactions imitate Table 6 lists those criteria employed in the authors'
RNase H activity. One is a combination of a DNase laboratory. No one criterion is absolute.
and RNase, a second is an exonuclease 111-like
activity which degrades both RNA and DNA 1. Biochemical Oiteria
moieties of hybrid structures, and a third is a a. Density
phosphodiesterase. In order to rule out these The intact type-C virus particle has a density of
pseudo-RNase H activities, it is important to assay about 1.16 glm1,"8 type-B particles about 1.18
the nuclease activities with several substrates, g / n - ~ l , * ~and
~ cores about 1.25 g/ml."8*'
for example: [3H] (rA), - (dT),, ['HI (rA)n, These densities are not unique to virus particles.
[3H] (U),, ['HI (dT), (rA),, and 4x174 DNA Some cellular membrane components may have
* [3H] RNA.'57>268If only a true RNase His similar densities. The density of virus particles also
measured, only ['HI (rA), - (dT), and 4x174 varies with many conditions such as the amount of
DNA * [3H] RNA are expected to release soluble membrane lipoprotein and cellular nucleic acids
oligoribonucleotides. In some studies, results with associated with the particles and the age of the
[ 3H](dT) *(rA), as substrate were not reported. particles. It can range from 1.13 to 1.19 g/ml. The
y3
Although HI (rA),*(dT), is commonly used as association of cellular components can be reduced
substrate, it is not the best for detecting low by repeated purification in equilibrium density
activity. 4x174 DNAS[~H] RNA and calf thymus gradients or by very mild treatment of the virus
DNA. ['HI RNA are more efficiently utilized than particles with nonionic detergent. Aging or
the synthetic substrates.' ' *' ' RNase H activity freezing and thawing tend to disrupt some viral
is sensitive to salt even as low as 0.1 M, and particles. The partially disrupted particles are
therefore its presence can be missed if care is not generally heavier than that of intact particles, since
taken to adjust salt concentrations when assays are the core fraction of the particle has a density of
made from samples eluting from columns. Another about 1.25 g/m1"8s"9 and the ribonucleo-
factor affecting the assay of this enzyme is protein complex is more than 1.3 g/m1.'209'21 In

314 CRC Critical Reviews in Biochemistry

 TABLE 6
Criteria for Reverse Transcriptase

Biochemical criteria
1. The enzyme activity is detected in a particle fraction with a density of 1.15-1.18 g/ml and shifted to above 1.25 g/ml
upon treatment with adequate concentration of nonionic detergent.
2. The endogenous reaction requires all four deoxyribonucleoside triphosphates. It is at least partially sensitive to RNase
and resistant to actinomycin D, and product analysis reveals that the DNA is hydrogen bonded to a large RNA and
covalently attached to a small RNA.
3. The purified enzyme is able to use (dG)oli (rC),, (dG)oli (rCm),, and (dT)oligo (rA), as prher-template,
but not (dT)o,igo (dA), and (?IT),. When unpurked enzyme is used, the (dT)oligo (rA), to (dT)oligo
(dA), ratio is high when Mg" or Mn is used as divalent cation.
4. The enzyme is capable of transcribing heteropolymeric portions of viral HMV RNA.
- -
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5. The enzyme activity is stimulated by nonionic detergent when (dT),ligo (rA),, (dT), (rA),,, activated DNA, or
-
(dG),ligo (rC), is used as prher-template.
6 . The size of the enzyme is about 70,000 daltons for mammalian type€ viruses, 160,000 daltons for most avian type€,
and 110,000 daltons for type-B virus or MPMV.

lmmunological criteria
The enzyme activity may be inhibited by purified IgG made against some known viral reverse transcriptase and not
significantly inhibited by IgG against cellular DNA polymerases.

these high density regions, the purity of viral reverse transcriptase, there is as yet no other
components increases. Therefore, the presence of known enzyme which catalyzes an endogenous
the enzyme activity in the core fraction prepared RNA-directed DNA synthesis.
by nonionic detergents is a useful criterion for
reverse transcriptase. The authors generally have c. Synthetic Rimertemplate Specificity
found intracellular reverse transcriptase in the Among many synthetic DNA. RNA hybrid
cytoplasm in the post-mitochondria1 and mito- primer-templates, (dT)oligo *(rA)n and (dG)oli,o*
For personal use only.

chondrial particulate fraction? 39-1 The (rC), are probably the most efricient for reverse
density has varied and probably depends on many transcriptase. Since purified reverse transcriptase
factors, especially the method of cell disruption, does not use (dT)oligo .(dA), while some cellular
but generally it has been between 1.13 and 1.19 DNA polymerases do, the authors of this article
Sld. routinely compare the utilization of (dT)o,igo~
(rA), with that of (dT)oligo*(dA)n. Though
6.RNA-directed Reaction cellular DNA polymerase y' 5 6 and 0' '
use
The first step to demonstrate that a given .(dT)oligo*(rA)n as template, they can also use
enzyme catalyzes RNA-directed DNA synthesis (dT)oli ;(dA),. Therefore, a high ratio between
reaction is to show that all four deoxyribonucleo- the uthzation of (dT)oligo.(rA)n to the utiliza-
side triphosphates, namely dATP, dGTP, dCTP, tion of (dT)oligo.(dA)n is an additional criterion
and dTTP, are required for optimal DNA synthesis for the presence of viral reverse transcriptase. This
in an endogenous reaction. Omission of one or ratio should be measured both with Mn" and with
more triphosphate should then reduce the amount Mg" as divalent cation. If Mg" is used as divalent
of DNA synthesized. One can rule out that the cation, a slight contamination with cellular enzyme
polymerization of deoxyribonucleoside mono- can easily reverse the ratio, since cellular poly-
phosphate is not due to terminal transferase by -
merases use (dT)oligo (dA), much more effi-
using only one of the nucleoside triphosphates. ciently than reverse transcriptase. The use of
Another conventionally used criterion is the (dT), *(rA), as primer-template to detect reverse
RNase sensitivity. As pointed out previously, this transcriptase activity' is not valid bacause
is not an absolute criterion and this test should be some cellular enzymes (both prokaryote and
performed in the presence of high salt and eukaryote) use it very well.9 y2 r2 '
relatively low concentration of RNase. However, Another useful criterion to distinguish cellular
definite proof that the reaction is RNA-directed DNA polymerases, particularly DNA polymerase
rests on the product analysis (see Section IIB2). y from reverse transcriptase, is that cellular DNA
Although a proper product analysis in itself does polymerases are able to synthesize (rA) using
not necessarily mean that the activity is from viral (rA)oligo (dT), as primer-template' ''
wMe

December 1975 315

 none of the viral reverse transcriptase tested portions of mRNA even if high concentrations of
is able to use this primer-tempiate (M. these enzymes are used.'
Robert-Guroff, personal communication).
(dG)o,igo-(rC), was thought to be completely e. Stimulation by Nonionic Detergent
specific for viral reverse transcriptase. However, Thompson et al. observed that reverse tran-
recently it was'shown that purified cellular DNA scriptase activity is enhanced by nonionic
polymerase 7 can use this primer-template albeit detergent."' This is not due simply.to protection
with poor efficiency.'58,284 However, since all from a nonspecific loss of enzyme, but rather is
reverse transcriptases use this primer-template due to an enhancement of the availability of some
efficiently, the authors use it as an important primer-templates which may have a tendency to
criterion, although again not an absolute one. collapse in solution, for example, (dT)oligo*(rA),,
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Recently, Gerard et al. reported that (dC)pligo. (dG),ligo..(rC), and activated DNA. This stimula-
(rCm), is specific for viral reverse transcriptase tory activity by nonionic detergents may be
both from type-C207 and type B particles.28S unique to reverse transcriptase. The activity of
Utilization of (dG)oligo*(rCm)n should, therefore, cellular DNA polymerase was not enhanced by the
be a useful criterion for distinguishing viral tran- presence of nonionic detergent. This could be an
scriptase from cellular polymerase. We confirmed additional criterion to help distinguish reverse
this observation. transcriptase from other cellular enzymes.

d. Natural RNA Primer-template f. Size

The authors think the most specific primer- As discussed in Section II.C.3 reverse tran-
template is the HMW RNA from viruses. Detection scriptase can be divided into three types based on
of DNA.RNA hybrids in a short-term reaction their molecular size. Among them the mole-
with this RNA is a direct demonstration that a cular weight of the subunit structure is unique for
given enzyme is capable of using the HMW RNA as each type of the enzyme. Since the enzyme tends
For personal use only.

primer-template. This is usually done by analyzing to aggregate in a low salt condition, an estimate of
the products in a cesium sulphate density equili- the molecular weight should be performed in a
brium gradient or by measuring the resistance of high salt condition or in the presence of detergent.
product-primer-template complex to S1 nuclease. The finding of an appropriate molecular weight for
Since viral HMW RNA contain a tract of (rA)n, it an enzyme in question is a helpful criterion in
is necessary to demonstrate further that the diagnosing it as reverse transcriptase.
products are not (dT), and are complementary to
heteropolymeric portions of the HMW RNA. 2. Immunological Criteria
Reduction of DNA synthesis following omission of As previously discussed, reverse transcriptase
one or more nucleotides and the demonstration of can be subdivided into six groups according to
DNA synthesis by using labeled deoxyribonucleo- their immunological properties. Inhibition of poly-
side triphosphate other than dTTP are some merase activity by IgG to reverse transcriptase
indications of transcription of non-(rA), regions. from a known RNA tumor virus indicates a
More definitive evidence can be obtained by relationship exists between the polymerases.
hybridizing the DNA products back to HMW RNA Generally, antibody to viral reverse transcriptase
in the presence of a relatively high concentration does not neutralize cellular DNA polymerases'
''
3

of (rA)". Although purified E. coli polymerase I 9'

''
and vice versa' y 3 . Therefore, lack of
transcribes HMW RNA,13 the amount of E. coli inhibition by antibodies to cellular DNA poly-
DNA polymerase I required for this transcription merases is a useful indication of the presence of
is about 200-fold more than that of reverse reverse transcriptase.
transcriptase. To date, eukaryotic DNA poly-
merases (a,p, 2nd r) have not been shown to 111. INTRACELLULAR
transcribe viral RNA. Therefore, transcription REVERSE TRANSCRIPTASES
of viral HMW RNA may be probably the most
specific criterion. Following the initial finding of a reverse
The use of globin mRNA in the presence of transcriptase-like activity in human leukemic cells
(dT)oligo is another useful criterion for reverse by Gallo et al.,' many studies have been carried
transcriptase since cellular DNA polymerases out to detect a "virus-specific" enzyme in a variety
apparently may not transcribe heteropolymeric of normal and malignant tissues and in virus
316 CRC Critical Reviews in Biochemistry
 infected and uninfected cells (Table 1). The Table 7. To prevent confusion, the old nomen-
purpose of these studies includes: (1) to obtain clature for these enzymes is listed in Table 8.
evidence for the presence of an RNA tumor virus For recent detailed reviews see Reference 289.
in certain neoplastic cells of man; (2) to under- DNA polymerase a is found in the cytoplasmic
stand the mechanism of viral replication in viral fraction of many tissues including human leuko-
and transformed cells (3) to elucidate the role, cytes and cultured c e l l s ~ 9 ~ 2 0 6 ~ 2 8 3 ~rat2 9 0 ,
liver 2 9 5-2 9 7 rabbit and mouse cells:89
if any, of reverse transcriptase in development 92993300

and differentiation; (4) to determine if detection Morris hepatoma:” rat ascites hepatoma
of the activity is useful for diagnostic and prog- cells,3023 3 0 3 calf t h y m u ~ , 3 ~and ~ >chicken
~ ~ ~
nostic modalities in human malignant diseases; and embryonic cells.’ ” 3 3 0 The amounts of this
(5) to study the relationship between cellular enzyme increase when cells are in a proliferative
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DNA polymerases and viral reverse transcriptase. stage.’ 7 4 y 2 9 9 3 0 0 Presumably then, this enzyme
In many of these studies, strict criteria for reverse is required for cell proliferation. DNA polymerase
transcriptase were not applied and therefore there a has been called the high molecular weight DNA
was confusion between authentic viral enzymes polymerase; it sediments a t 6 to 8s. The reported
and some cellular enzymes. Reverse transcriptase molecular weight ranges from 90,000 to 250,000
from human leukemic cells is the only well-studied daltons.’ ,2 ‘9’ 3’ *3 The high molecular
intracellular reverse transcriptase‘ y9 described to weight form probably represents an aggregate
date from cells not known to be producing virus. form.
It is, of course, easier to isolate viral reverse Recently, Yoshida et al. have found inter-
t r a n s c r i p t a s e f r o m v i r u s - p r o d u cing convertible molecular species of cytoplasmic DNA
cells.99 , 1 0 0 9 4 2 1 polymerase from calf thy mu^.^ O 4 Since nucleic
Reverse transcriptase has been identified in two acids were not removed prior to fractionation by
locations in cells, the cytoplasm and the cisternae. gel filtration, the results may be explained by
The enzyme in the cytoplasm is associated with association of enzyme with nucleic acids. The
For personal use only.

the post-mitochondria1 pellet fraction and in basic subunit of these aggregates has a molecular
“A” particles is found in cisternae. The relation- weight of 90,000,304which agrees with the recent
ship, if any, between these two reverse trans- findings of Sedwick et Smith et al.,”’
criptases from these two particulate fractions and Chang et al. (personal communication).
is not known. For convenience, the reverse tran- In general, the preferred divalent cation is Mg”
scriptase associated with these two particulate and the preferred primer-templates are DNAs such
fractions will be discussed separately. as activated DNA and (dA-dT),. The enzyme does
It is obviously much more tedious to obtain not use (dG)oligo-(rCln, natural RNAs, or mito-
purified reverse transcriptase from cells than it is chondrial DNA as primer-template. In the presence
from virus. This is due, among other things, to the of Mg”, DNA polymerase a! uses (dT);(A), and
presence of cellular DNA polymerases (at least (dT)oli,o-(dA),. (dT)o,igo-(rA)n is used with
one which has properties similar to those of reverse very poor efficiency if at all. This enzyme is
transcriptase), the distribution of reverse tran- relatively sensitive to N-ethylmaleimide (Ki=lO -’
scriptase in various subcellular compartments, the M)’ 0 6 , 2 8 3 $28 9 , 2 9 3 and to salt.Z06 ,309 Th e PH
low amount of reverse transcriptase per unit of optimum of this enzyme is 7.5 to 8.296Recently,
protein (in the case of nonproducer cells), and the Smith et al. prepared antibodies (in rats) to DNA
presence of nucleases and proteases. In order to polymerase a isolated from human lympho-
distinguish reverse transcriptase from cellular DNA cytes.’ ” The purified IgC does not inhibit the
polymerases, the major properties of the cellular activity of DNA polymerase 0 from the same
enzymes must be known. They are summarized cells or different types of cells.’ 5 7 Chang
below. and Bollum3I0 originally found a partial cross
reaction of polymerase (Y and 0 with antisera
A. Eukaryotic Cellular DNA Polymerases prepared in rabbits. However, in agreement with
Four distinct cellular polymerases have been Smith et al. and Weissbach et Bollum
isolated from a variety of mammalian cells. They with a different antiserum (personal communi-
are DNA polymerase a, DNA polymerase 0, DNA cation), more recently did not find the cross
polymerase 7, and mitochondria1 DNA poly- reaction. The original antigen of Chang and
merases. Some relevant biochemicai and immuno- Bollum must either have been contaminated
logical properties of these enzymes are listed in with DNA polymerase p or more likely the anti-
December 1975 317
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For personal use only.

w TABLE 7
L
00

Some Biochemical Propexties of Various Cellular DNA Polymerases

Human leukemia and

DNA Polymerase DNA Polymerase DNA Polymerase Mitochondria1 mammalian viral
Properties 0 P 7 DNA polymerase reverse transcriptase
a Cellular location Cytoplasm Nucleus and/or Nucleus and/or Mitochondria Cytoplasm
1
E' cytoplasm cytoplasm
F, Molecular weight* 0.9 0.4 1.o 1.0 0.7
3 (Daltons X lo-') (6-8s) (3.5s) (6.1-6.3s) (45s)
Utilization of
Templateprimer * *
Activated DNA
(dT),bgo * (dA)n

-
(dG),,igo - (a" -
(rA)oligo (dT), N.R.
70s RNA N.R.***
Mitochondria DNA (Mg")
Nonionic detergent
stimulation no no no
- N.R.
N-eth ylmaleimide
inhibition Sensitive Insensitive Intermediate Insensitive Sensitive

*The value from the smallest subunit reported is used.

**++I+: High efficiency; ++ - *: moderate utilization; +: slight utilization; i: marginal utilization; and -: not used.
***N.R.: Not reported.
 TABLE 8
Summary of Synonym of Eukaryotic Cellular DNA Polymerase

a* o* Y* Reference

1 I1 111 Smith and Gallo’

Lewis et al.’
D-I1 D-I R-I or R 11 Weissbach et al.’
Fridlender et al.’ I
Spadari and Weissbach’ 5 8
C or N2 N1 Sedwick et al.’
High molecular Low molecular Chang and Bollurn’ *
weight weight Chang and Bollurn’
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(6-8s) (3.39 Coleman et al.’ O

(maxi) (mini)

*See Reference 110.

genic determinant t o which the host animal from chicken cells.222 This antibody is able t o
responded was more broadly reactive. The IgG to neutralize the activities o f avian DNA polymerase
DNA polymerase (Y also does not inhibit DNA a and /3 from various strains of chickens and rat
polymerase y I s 6 nor any of the virus-related cells, DNA polymerases of REV-T and TD-SNV,
reverse transcriptases.8>9,1 ’
This antigenic and rat DNA polymerase /3, but not rat DNA
property can therefore be used t o distinguish DNA polymerase a. Many of the biochemical and
polymerase (Y from reverse transcriptases and immunologjcal properties of DNA polymerase /3
probably other cellular DNA polymerases. make it easy t o distinguish it from reverse
DNA polymerase /3 has been found in the transcriptase.
For personal use only.

cytoplasm and in the nucleus of a variety of DNA polymerase y is found in the nucleus and
tissues. These reports include human leukocytes sometimes in the cytoplasm. Some of its
and cultured cells,g ,’ O 6 3’ ,’ 92 ,’ rat properties are similar t o those of reverse transcrip-
liver ,’ 5 - 2 ’’
r a b b i t tissues,’ ,’ a calf tase, and therefore a thorough familiarity with the
thymus,3 ’ ’
93 murine tissues,’ 9 9 9 3 0 0 rat ascites properties of this enzyme is advisable when
h e pat o m a tissue^,^ 33 ’ Morris hepatoma examining cells for reverse transcriptase. This
’
tissue,30 and chicken embryonic cells.’ ”,3 O ’ enzyme, in fact, was probably the activity
DNA polymerase p has a molecular weight of detected earlier by a number of workers in various
about 40,000 t o 50,000 daltons. It sediments at cells and confused with reverse transcriptase. It
about 3.5s. Activated DNA and (dA-dT), are the was first named R-DNA polymerase due t o its
most efficient primer-templates for this enzyme in ability to copy the RNA strand of synthetic
the presence of Mg”, while (dT),,-(dA), and RNAaDNA homopolymer hybrids.’ Since both
(dT), -(rA), are the best primer-templates in the L cells and HeLa cells used in these studies might
presence of Mn++. (dG)oligo.(rC)n, HMW RNA, have been producing low titers of a RNA tumor
and mitochondria1 DNA are not utilized. Several virus, the evidence that this was a new polymerase
factors affect the template specificity of the was not final. However, the presence of this
enzyme, for example, the purity of the enzyme, DNA polymerase as a distinct enzyme was verified
divalent cation, and salt concentration. The when Lewis et a1.99y’56and McCaffrey e t d.*”
enzyme activity is stimulated by high ionic isolated this enzyme from human normal and
strength. The optimal salt concentration is leukemic leukocytes and Evans et al.’” from
between 0.1 M and 0.2 M NaCl. The pH optimum mouse spleen. These studies demonstrated that
of the enzyme is 8.5 to 9 . 0 . 2 8 9 The enzyme DNA polymerase 7 is distinct from DNA poly-
activity is not stimulated by nonionic deter- merase (Y and p, and also from viral reverse trans-
gent.lZ4 It is not inhibited by NEM2069290 b Y criptase.’’ This was further confirmed when
antibodies t o DNA polymerase a,’ 9 3 nor by ’ ’ Spadari and Weissboch purified this enzyme.’ ”
antibodies to viral reverse transcriptase.’ >g The DNA polymerase y in our studies has a molecular
only antibody t o DNA polymerase /3 available is weight of approximately 100,000. It is able t o

December 1975 319

 copy activated DNA, (dT)oligo (dA),, and (dA- on the antigenic relationships of this enzyme to
dT), with moderate efficiency. It also efficiently other DNA polymerases. However, this enzyme is
uses (dT)oligo (rA), and (dT), (rA), in the easily distinguished from reverse transcriptase by
presence of Mn". However,with Mg*,it isvirtually many criteria.
inactive with these primer-templates. The optimal
salt concentration for DNA polymerase y is
B. Intracytoplasmic Reverse Transcriptase
about 100 mM. If the salt concentration is de-
From virus-producing cells, i t is a relatively
creased the capacity to use (dT)oligo (rA), is simple task to isolate and purify virus-related
diminished while ability to use (dT)oligo .(dA), is reverse transcriptase. However, from the cells not
enhanced. The authors believe an important
producing virus, if present at all, its detection
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criterion in distinguishing polymerase y from and isolation is very difficult. Intracytoplasmic

reverse transcriptase is to determine the activity
reverse transcriptase is usually found in the high
with (dT)oligo-(dA)n with Mg" and Mn". Poly- speed (90,000 xg) particulate fraction from
merase y will use (dT)oligo.(dA), in Mg", but the post-mitochondria1 fraction. This particulate
under no circumstances does reverse transcriptase fraction has provided the most successful method
use (dT)oligo-(dA)n in the authors' experience. to detect and isolate reverse transcriptase for the
Recently Spadari and Weissbach reported that the authors of this article. An extensive study of the
polymerase y from HeLa cytoplasm could be
nature of the cytoplasmic particulate fraction has
further fractionated into two peak activities in
been carried out with human leukemic blood
b o t h p h o sphocellulose and hydroxylapatite
cells." This can serve as a model system for the
columns.' ' One peak, y l , is able t o transcribe
study of intracellular virus-like particles and
(d T 10 1 i g 0 * (r A 1n but not (dG)oligo * ( c > n
9
associated reverse transcriptase.
(dC)oligo*(I)n and (dA)oligo.(U)n. However, the
other peak, 72, is able to transcribe all of these
primer-templates with relatively high efficiency. I . Reverse Transcriptase from Human Leukemic
For personal use only.

The first peak, y l , is similar to the DNA poly- Cells

merase y found by Lewis et a1.Is6 The second Initially, reverse transcriptase-like activity was
peak, y2, is a new activity. Apparently, both observed in three cases of acute leukemia but not
enzymes are antigenically unrelated to reverse in PHA stimulated lymphocytes.' The activity was
transcriptase isolated from SSV-1, MPMV, and detected in nucleic acid-free "cytoplasmic"
R-MULV.' supernatant, but subsequently, the high speed
Mitchondrial DNA polymerase has been cytoplasmic pellet fraction was identified as the
isolated and purified from mitochondria of HeLa '
best subcellular fraction.6 73 * DNA synthesis can
cells' O 6 ,'' and rat liver.' ,3 ,' ' ' The be obtained in the cytoplasmic pellet with endo-
molecular weight of the enzyme is about 100,000 genous primer-template. The reaction is sensitive
which is significantly larger than that of DNA to RNase and the DNA product bands as an
polymerase 0, but similar to those of DNA poly- RNA-DNA hybrid. The enzyme purified from this
merases a and y. This enzyme can also efficiently fraction is able to transcribe viral RNA? and in
9'

use activated DNA as template-primer. However, at least some cases of adult AML, is is antigenically
mitochondrial DNA polymerase is distinguished related to reverse transcriptase from certain primate
from the other DNA polymerases by the fact type-C viruses."' The biochemical studies were
that it can use native mitochondrial circular extended to all types of leukemia: but in our
DNA as template-primer (initiation of nick studies not all leukemic cases were found to contain
by an endonuclease). Mitchondrial DNA poly- reverse transcriptase. To date, it is only with AML
merase does not copy (dT)oligo.(rA),, (dT)oligo cells that the immunological relatedness to known
(dA)n (dA)oljgo*(rWn, (dc)oljgo.(Gn
9 and viral reverse transcriptase has been found. The
(dC)oligo*(rI)n.306 This is in contrast to DNA reproducibility of detection has been improved by
polymerase y which uses all of these template- banding the cytoplasmic pellet fraction in sucrose
primers to varying degrees. Similar to DNA poly- density gradients and isolating the particulate
merase p, the mitchondrial polymerase is relatively region from a density of around 1.16 to 1.18
insensitive to NEM.306 There are no studies dml.' The specific activity of the enzyme can be
regarding the utilization of natural RNA nor any enhanced by repeated banding in a sucrose density

320 CRC Critical Reviews in Biochemistry

 gradient.’ With this modified procedure, in some with a nonionic detergent. This density is similar
cases it is possible to detect reverse transcriptase to that of the virus cores. The size of the 1.16
activity with about 109 nucleated cells. It is g/ml particles varies from 500s and 1,000s and
important to emphasize that with this relatively sometimes they form larger aggregates as judged
small amount of leukemic cells, one has to isolate by gel filtration.’ ’ Virus-related nucleic acid
the cytoplasmic pellet in order to detect reverse (RNA) and virus-related reverse transcriptase are
transcriptase activity, and it has not been possible complexed in these particles. Like RNA tumor
to detect this enzyme activity by isolating it from viruses they give rise to an endogenous DNA
“nucleic acid free” cytosol” (B. Lewis and R. C. synthesis in the test tube. Most of this reaction is
Gallo, unpublished observation). In our experience, resistant to actinomycin D, but it is sensitive to
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reverse transcriptase is detectable in only 20% RNase to some degree. The degree of resistance to
to 30% of patients with leukemia. Moreover, actinomycin D and sensitivity to RNase varies
we emphasize again that it is only with adult from case to case and from preparation to prepara-
AhK that the immunological relationship to tion. In general, a better result is obtained when
known primate type-C viruses has been demon- the particles are purified more extensively by
strated so far, and even in adult Ah4L, only about repeated banding.” The products from the endo-
one-third of all patients exanlined have detectable genous reaction are covalently linked to an RNA
enzyme. This is in contrast to the data of primer and associated with HMW RNA through
Spiegelman and colleagues who have reported hydrogen bonds.6 Similar to the products of a
detection of reverse transcriptase in virtually all viral endogenous reaction, the DNA products are
cases of leukemia examined.’ small but contain sequences complementary to
RNA of some known type-C primate oncogenic
viruses (SSV-I and GaLV” and R-MuLVI3).
a. Purification
Additional evidence that the cytoplasmic particles
The procedures described here are those that
contain virus-related RNA is based on positive
For personal use only.

isolated the cytoplasmic particulate fraction

molecular hybridization between particle RNA
before solubilization and purification of reverse
and DNA products from known type€ primate
transcriptase.
oncogenic viruses. The size of the virus-related
RNA in the particle is not known with certainty,
(1) Cytoplasmic Particles but the presence of HMW RNA is suggested by a
To prepare the cytoplasmic particulate fraction, positive “simultaneous detection” assay,’ ’’
fresh human leukemic cells are mechanically dis- and more recently by direct labeling of the
rupted. The nucleic acid and mitochondria are RNA.’ 7 8 There is evidence indicating that reverse
removed by a differential centrifugation at 1,000 transcriptase isolated from these particles is
a n d 1 2 , 0 0 0 x g , respectively. The post- viral-related. This will be discussed below.
mitochondrial supernatant is further centrifuged at
a high speed (90,000 xg) to obtain the post- (2) Solubilization of Reverse Transcriptase
mitochondrial pellet fraction. The pellet fraction is The advantages of isolating intracellular reverse
then banded from one to three times in an transcriptase from cytoplasmic particles are
equilibrium sucrose density gradient and the
RNase sensitive endogenous DNA synthesizing 1. It requires as little as lo9 cells to obtain a
activity is detected from fractions with a density reasonable amount of enzyme activity.
of approximately 1.15 to 1.19 g/ml. These 2. It is easier to obtain enzyme with relatively
fractions contain reverse transcriptase and nucleic good specific activity.
acids. To enhance the specific activity of the 3. One can obtain the presumed native RNA
endogenous reaction, the pellet fractions are template and prepare labeled DNA probes.
banded repeatedly in the sucrose density
’
gradients.’ These particles have some properties The enzyme can then be further purified by
similar to those of type-C RNA tumor viruses (see conventional purification procedures such as
Reference 320). For example, in some cases these DEAE cellulose and phosphocellulose chroma-
particles can be converted to an entity with a tography and Sephadex (3-200 gel filtration.6
density of approximately 1.25 d m l by treatment About a 200-fold purification was obtained over

December 1975 321

 that of the pellet fraction. Alternatively, the b. Evidence of Viral Gene Expression or Virus
enzyme can be purified by a Sepharose 4B gel Infection
filtration ' This procedure has been (1) General Criteria
especially effective with small amounts of cells. The presence of viral components in tumor cells
The solubilized and partially purified reverse might be indicative of a previous history of virus
transcriptases exhibit the biochemical and infection and/or expression of an endogenous viral
immunological properties of known murine and genome. Viral components which have been
primate viral reverse transcriptases (see Table 6 for investigated mast thoroughly in this regard are
the criteria of viral reverse transcriptase and recent nucleic acids, p30 protein, and reverse transcrip-
summary by Gallo' 0 9 ) . The enzyme significantly tase. They are summarized in Table 9. The
prefers (dTIoligo (dA), over (dTIoligo (W, presence of reverse transcriptase has been used as
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as primer-template and can efficiently copy evidence of viral gene expression in a variety of
(dG)o,jgo *(rC)n.6 ,9 These activities are stimulated human malignant tissues due to its relative sensi-
by nonionic detergent.' 2 4 Similar to many DNA tivity. However, as emphasized above, it is
polymerases, the human virus-like DNA poly- extremely important to examine the specificity of
merase can copy activated DNA, (dT), *(rA), and the "reverse transcriptase-like" enzyme detected in
(dA-dT)n.6 Another important feature of this these tissues. Reverse transcriptase from human
enzyme is its ability to transcribe the hetero- leukemic cells is the only intracellular reverse
polymeric portions of both avian and mammalian transcriptase which has been thoroughly studied.
viral HMW RNA.69' The molecular weight of Among the criteria of reverse transcriptase describ-
human reverse transcriptase was found to be ed in Table 6 , four are most pertinent: (1) high
130,000 when the enzyme was solubilized in low preference of (dT)oligo -(rA), over (dT)oljgo'
salt buffer.6 However, a low molecular weight (dA), as primer-template; (2) ability to efficiently
form with a molecular weight of 70,000 was transcribe (dG)oligo-(rC)n and (dG),,igo *(rCm)n;
obtained when the enzyme was solubilized in (3) ability to transcribe heteropolymeric portions
For personal use only.

the presence of high salt buffer and Triton of HMW RNA; and (4) antigenic relatedness to
~ - 1 0 0 . ~ The
9 ~ ~ two forms of enzyme are reverse transcriptase from known type-C viruses
interconvertible. The HMW forms can be convert- and absence of this relationship to the known
ed to LMW form by treatment with high salt and cellular enzymes.
nonionic detergent (presumably to remove lipoid
components) and LMW form can be reaggregated (2) Immunological Relationships
to the HMW form by a dialysis against a low salt The immunological relationships of the intra-
buffer.' Primer molecules are required for cellular enzyme t o known RNA tumor viruses may
this reaggregation. These two forms of the provide information useful for developing diagnos-
enzyme exhibit some variant biochemical and tic or prognostic tests. For example, one might use
immunological properties. These are (1) the HMW this property to develop in situ irnmunoprecipita-
form utilizes synthetic primer-templates less tion techniques. This may be especially valuable
efficiently than the LMW form: (2) the HMW form for human tissues which do not produce detect-
transcribes HMW RNA in the absence of synthetic able extracellular particles. Table 10 shows a
primer relatively more efficiently than the LMW summary of the immunological relationships of
form of reverse transcriptases.' ' '
9' O (3) the HMW reverse transcriptases from the primate viruses and
form is not inhibited by the antibody to primate from leukemic cells. From these antigenic relation-
oncogenic viral DNA polymerase. This variation in ships, the primate viruses can be divided into three
the enzyme following conversion of molecular distinct groups: the t y p e 4 frankly tumorigenic
weight may have some functional significance in virus group, the type-C endogenous virus group,
v i m . Apparently, this phenomenon is not unique and the MPMV virus. AU three groups are not
to human intracellular reverse transcriptase, since detectably related to human cellular DNA poly-
it has also been observed in the intracellular merases a, p, and 7.The reverse transcriptase from
transcriptase isolated from lymphoblasts deliber- some human AML blood cells is closely related to
ately infected by and producing GaLV,' murine ' the polymerase from type-C infectious oncogenic
cells producing R-MuLV,' ' and the extracellular viruses. This information may be useful for further
viral reverse transcriptase from R-MuLV (Refer- exploration and understanding of the viral related
ence 201 and unpublished observation). information in human cells.
322 CRC Critical Reviews in Biochemistry
 TABLE 9.
RNA Virus Related Information in Human Tumor Cells

Type€ animal virus probe

RNA tumor virus-like component

searched for in human cells Primate Murine Avian

1. Virus-like DNA in genome N.D. +3 2 3 -3 1 3

2. Virus-like DNA (product of re- +++I + I 1 ,I 3 - 12,l I

verse transcriptase synthesized

endogenously “in vitro”)
3. Viral-like RNA in cytoplasm +or+++!’ +’ 6 - I2,16
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4. Viral-like reverse transcrip +++6,9>10* +++6,9 ,I 0 8 +6 ,9 ,I 0 8

tase biochemical evidence

5. Virus-like reverse transcriptase- +++a 99 +a 99 -8
immunological evidence
6. Virus-related antigen (p30) -3 I1 +,I1 -3 1 1
7. Virus-like organelle (cytoplasmic +++I 1,13 +I I ,I 3 -I 2
particle with density of 1.16 to
1.17, containing reverse trans-
criptase and virus-like RNA in a
functional complex)

+++: strong similarity, approaching identity: +: weak, but detectable similarity;

- : no detectable similarity; N.D.: not yet detected, still under investigation.

TABLE 10
Immunological Relatedness of DNA Polymerase from the Known Primate Viruses
For personal use only.

and Cellular DNA Polymerase*

Antibody to DNA polymerase of

DNA Polymerase from SSV-GALV M-7 MPMV Human leukocytes

SSVGALV +++ 0 0 0
Human AML cells, HL23 +++ 0 0 0
virus, Hep 1 2 virus,
and C-MuV
M-7 (Baboon 0 +++ 0 0
endogenous virus)
MPMV 0 0 +++ 0
DNA Polymerase a 0 0 0 +++
*+++: More than 90% enzyme activity inhibited by I&; 0: No inhibition

Other Relate nzymes (dT)oligo.(dA)n but not (dG)oli o*(rC)n, while

To establish authenticity of intracellular reverse reverse transcriptase can transcrbe (dT)oligo
transcriptase, several enzyme activities should be ( r N n and (dGloligo (rC),* but not (dT)olig(,
ruled out, for example, cellular DNA polymerase 7 (dA),. DNA polymerase 0 does not transcribe
and 0,’5 6 , ’ 5 0 , 2 1 6 terminal transferase,”
26
’25 ’
33 HMW FWA; it is smaller in size than reverse trans-
and an RNA-dependent but DNA-directed criptase, is insensitive to NEM, and is immuno-
endogenous DNA synthesizing activity.’ O 3 7 1 ’ logically unrelated to reverse transcriptase. DNA
172,327
These can be distinguished by a combined polymerase 7 can be distinguished from the viral
use of primer-template specificity, product analy- enzyme by size (it is larger), by its inability to
sis, and immunological examination. For example, copy viral HMW RNA, and by immunological
in the presence of Mn*, the purified cellular DNA studies. Terminal transferase simply can be ruled
polymerase 0 can transcribe (dT)oligo-(rA)n, and out by observing a reduction in DNA synthesizing

December 1975 323

 activity with the omission of one or two nucleo- eluted at 0.45 M KCl. The 0.3 M KC1 DEAE eluate
side triphosphates and with the lack of DNA contains DNA polymerase a and 7. These two
synthesizing activity in the presence of prinier enzymes can be fractionated further either by a
alone which the transferase utilizes. Also it is DNA cellulose column or a hydroxylapatite
much smaller in size than the viral enzyme. The column. In the DNA cellulose column, DNA
RNA-dependent DNA-directed DNA synthesizing polymerase a is eluted at 0.14 M KCl, while DNA
activity is observed in many normal tissues' ' polymerase 7 is eluted at 0.2 M KCl. The purity of
and also transformed cells.3 27 The distinction of the reverse transcriptase was not determined.
this activity from RNA-directed DNA polymerase Reverse transcriptase purified by this method has
depends on careful product analysis by demon- both biochemical and immunological properties
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strating the lack of DNA-RNA hybrids in newly closely related (if not identical) to that isolated
prepared DNA products (Table 2), by immuno- from extracellular viruses. When a similar scheme
logical examination, and by enzyme purification. was used to fractionate DNA polymerase from
It is not known which cellular enzyme is PHA stimulated human lymphocytes and from
responsible for this reaction. human leukemic cells, all DNA polymerase a, 0,
and 7 were obtained but no reverse transcriptase
was detected." 9' ' This finding presents a
2. Reverse Transcriptase from Cells Producing dilemma regarding the usefulness of this procedure
RNA Viruses for the isolation of viral enzymes from nonproduc-
Intracellular reverse transcriptase has been ing cells. Perhaps, the cells used in the study did
isolated from various cells producing RNA viruses not contain reverse transcriptase or contained too
such as Bal b/3T3 cells producing (Ki-MuLV4' ' little of the enzyme for it to be detected. On the
or M - M U S V ~mouse
~ ~ spleen cells infected by other hand, reverse transcriptase is greatly enrich-
R-MuLV,' O 0 NC37 cells infected by and produc- ed in the microsomal pellet fraction. In fact, Yang
ing SiSV-199 or GaLV", IdU induced Balb- et al.'O0 and others6)318 found that most of the
For personal use only.

K/3T3 cells ' O 2 and mainmalian cells (human, intracellular reverse transcriptase is located in the
dog and mink) chronically producing RD114 post-mitochondrial microsomal fraction. The
v i r ~ s 4 ~ 'The enzymes are isolated either enzymes in the pellet fraction were solubilized in
from cell homogenates' ,' ',2 y4 ' or from high salt and nonionic detergent and purified by
the inicrosoinal pellet fraction.' '33 * repeated hydroxylapatite chromatography'
One difficulty in using whole cell or cytoplasmic and/or gel filtration.' J O 0 The enzyme thus
homogenates is separating cellular DNA poly- obtained is approximately 800 to 1400-fold
merases from reverse transcriptase. In most studies enriched and has most of the biochemical and
with this procedure, the purity of the enzyme is immunological properties of viral reverse transcrip-
not critical. Recently, Lewis et al." described a tase. This procedure is particularly useful in
method to isolate and separate cellular DNA detecting minute amounts of intracellular reverse
polymerase a, 0, and 7 and reverse transcriptase. In transcriptase from cells containing viral informa-
this method, cells are homogenized, enzymes are tion but not producing virus particles (see below)
extracted with 1 M KCl and nonionic detergent, including human leukemic cells. Another method
nucleic acids are removed by fibrous DEAE to isolate reverse transcriptase from infected cells
cellulose, and the enzymes are fractionated on is to use affinity chromatographic column as
microgranular DEAE cellulose by eluting with 0.05 exampled by a recent report by Gerwin et al.
M and 0.3 M KCI, respectively. It is interesting to By using (dT)o,igo cellulose column, they have
point out that if the buffer contains Triton X-100, purified RD114 virus DNA polymerase in one
almsot all viral enzymes were eluted from the step chromatography and found that the molecular
column at 0.05 M KCl, but if the buffer does not weight of intracellulw reverse transcriptase of
have Triton X-100, only a portion of the enzyme RD114 virus was 95,000. This is in contrast to
was eluted from the column at 0.05 M KCI. Viral reverse transcriptase of purified RD114 virus that
enzyme and DNA polymerase 0 eluted by 0.05 M has a molecular weight of 70,000. In this study,
KCl are fractionated fu,rther on a phosphocellulose no aggregate form was observed. This is presumed
column with a KCl salt gradient. With this method, due to the nature of this particular affinity column
the viral enzyme is eluted at 0.2 M KCl and is or due to the fact that it contained Triton X-100
separated well from DNA polymerase 0 which is in their buffer.
324 CRC Critical Reviews in Biochemistry
 3. Is Reverse Transcriptase Present in Normal there is little evidence that the enzymes used RNA
Tissues? as primer-template. A demonstration that the
There are many reports regarding the presence purified enzyme has properties like known viral
of RNA-dependent DNA polymerase in normal reverse transcriptase and a demonstration of DNA
mammalian tissues. However, only one of these RNA hybrids in the endogenous reaction has not
studies has definitely demonstrated that the been reported. Bobrow et al. observed an RNase-
enzyme has the characteristics of viral RNA- sensitive endogenous DNA synthesizing activity in
directed DNA polymerase. This enzyme was the microsomal pellet fraction of PHA stimulated
obtained from the placentaembryo of normal lymphocytes.' " This activity was shown to be an
rhesus m ~ n k e y . ' ' ~ It has also been reported RNA-dependent DNA-directed DNA synthesizing
earlier that type-C virus can be seen in the pla- activity.' 7 2 The possible presence of a minute
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'
cental syncytial trophoblasts of rhesus m ~ n k e y . ~ amount of RNA-directed DNA polymerase has not
The methods used in this study are as follows: been definitely ruled out. The relationship of this
The post-mitochondria1 microsomal fraction was activity, if any, to reverse transcriptase activity
obtained from a rhesus monkey placenta. The may be important in determining if reverse tran-
enzyme was solubilized by high salt in the presence scriptase plays a role in normal cellular prolifera-
of nonionic detergent and processed sequentially tion and differentiation.
through a DEAE cellulose and phosphocellulose
column. The enzyme thus obtained had most 4. Reverse Transcriptase Activity in Cultured
of the biochemical properties of mammalian typeC Murine Cells
viius enzymes, but antigenically it was closely A new DNA polymerase activity termed Peak A
related to reverse transcriptase from M7 virus, was isolated from the cytoplasmic pellet fraction
an isolate obtained through cocultivation of of two cultured murine cell lines.' One is from
baboon placental cells with heterologous c e k 6 ' a clone of BALB/3T3 contact inhibited fibroblasts
This virus is now known to be an endogenous called A31 and the other is from a subclone of
For personal use only.

virus of baboons. Therefore, reverse transcriptase nonproducers obtained by infecting A3 1 with

isolated from placenta is probably a gene product Ki-MuSV termed KA31. Neither cell line pro-
of the endogenous type-C virus of primates and duces virus. Peak A is eluted at approximately
differs from the known oncogenic and infectious 0.22 M KC1 in a phosphocellulose column with a
primate type-C viruses. It has also been suggested salt gradient. The amount of Peak A activity from
that chicken embryo contains reverse transcriptase A31 and KA31 was comparable although KA31
activity. This is based on three observations: cells might have a partial expression of the
(1) the endogenous reaction is sensitive to sarcoma genome3 and may also contain type-A
RNase;lo4 ( 2 ) DNA RNA hybrids are formed particles."' Peak A activity is similar to viral
in a short-term endogenous reaction: ' (3) DNA reverse transcriptase in several ways: (1) the
products are homologous to some RNA isolated chromatographic pattern is nearly identical. For
from the cytoplasmic pellet fraction.'04 It will be example, if similar purification procedures were
of interest to learn the biochemical and sero- applied to the clone of spontaneously transformed
logical properties of the purified enzyme. cells continuously producing virus, termed SzC13,
In some studies, activity with (dT), * (rA), or to extracellular R-MuLV, reverse transcriptase
was used as evidence for reverse transcriptase in would be eluted at the same KCl molarity; (2) the
normal cells,' $' 4 8 but as discussed above it is molecular weight is 70,000 which is the same for
now known that at least two cellular DNA reverse transcriptases from mammalian type€
polymerases can also efficiently use (dT), (A),. viruses; (3) the enzyme can efficiently use
These activities were almost certainly due to (dT)oli (rA), as primer-template; and (4)
polymerase 7. A RNase-sensitive endogenous re- anti-R-huLV IgG neutralizes 30% of Peak A
action was reported with normal rat tissue culture activity but this is less inhibition than is found
cells.'03 In this study, the presence of DNA * with authentic purified R-MuLV reverse tran-
RNA hybrids was not reported, although small scriptase. It is not known whether this lack of
fractions of DNA products were shown to hybrid- complete neutralization activity is due to a con-
ize to RNA isolated from the pellet fraction. tamination by other cellular enzymes, due to
However, it is still possible that the RNA used for molecular aggregation," or that it is simply a
hybridization is the transcriptional product from different enzyme. The only distinct difference
DNA existing in the pellet fraction and therefore between Peak A activity and authentic reverse
December 1975 325
 transcriptase is that Peak A activity is not able to particles performed in the presence of actinomycin
transcribe the rC strand of (dG)oli * (a),or D shows that the DNA products are initially
HMW viral RNA and it is inhigited less by associated with RNA and 70 to 90% of the
antibody to viral reverse transcriptases. The failure purified DNA products can be back hybridized to
to transcribe 70s RNA and (C), does not HMW RNA of the A particles.'" These findings
appear to be due to the presence of an inhibitor. provide some suggestive evidence that DNA poly-
Peak A activity is not DNA polymerase 7 as judged merase in type-A particles is very similar t o that of
by chromatographic behavior, molecular weight, authentic reverse transcriptase. It will be of
and antigenic relatedness. The biological signifi- interest to determine if the polymerases of A
cance of Peak A activity is not known. It will be of particles and type4 viruses are immunologically
interest to learn if it represents the DNA poly- related.
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merase associated with intracytoplasmic A par-

ticles or whether it is a precursor of reverse IV. INHIBITORS OF VIRAL
transcriptase or even a degraded component of REVERSE TRANSCRIPTASE
reverse transcriptase.
A. Rationale
C. Intracisternal A-particle Associated Reverse A completely specific inhibitor of reverse tran-
Transcriptase scriptase is not yet available. Specificity is relative
Intracisternal A particles are frequently ob- and often varies with the system measured. For
served in tumors of many species from birds to example, a compound can have a Ki value less than
man. These particles are observed both in normal M with respect to reverse transcriptase but a
and neoplastic tissues33 y3 3 4 in mice. Their mode Ki of M to another cellular enzyme. The
of transmission is not known. It has been specu- specificity of this compound in a given biological
lated that intracisternal A particles represent some system then depends on the role of the other
sort of viral gene expression and might be an enzymes in affecting the viability of the'cells. If a
For personal use only.

immature type-C virus. However, the latter possi- small depression of the other enzyme's activity
bility is unlikely due to the fact that type-A lead to a serious cytotoxicity, this compound is
particles contain a group-specific protein which is naturally not specific in the system under study.
antigenically unrelated to that of type-C particles. Valuable specificity is obtained only when an
Recently, HMW RNA was found to be associated inhibitor exerts its effect on a particular function
'
with intracisternal A particles.' ,2 * s3 A de- in a biological system without inducing cyto-
termination of the relationship between this HMW toxicity. In searching for specific inhibitors, the
RNA and that of type4 virus has recently been enzymes chosen for comparison are important.
completed and does not support the idea that they Many studies have been done with either avian or
represent immature type-C virus.' O 7 mammalian viral reverse transcriptase, since these
The DNA polymerase associated with intra- two types of reverse transcriptases have some
cisternal A particles has been initially described by different characteristics. This should be taken into
Wilson and K ~ f f who ' ~ ~reported that it can only consideration when comparing results obtained
use (dT)oli - (rA), as primer-template. There
was no entogenous activity detectable, and non-
with the two systems. Many studies also claim
specificity (or selectivity) of a compound by
ionic detergent was not required for the activity. comparing an inhibitory activity of E. coli DNA
However, if A particles are carefully prepared, an polymerase to that of reverse transcriptase. This
endogenous RNAdependent DNA polymerase type of study is not relevant to the specificity of
activity, can, in fact, be detected.'06'' O 7 The the compound in higher organisms. Comparisons
endogenous reaction is stimulated by nonionic are needed between the effect on reverse tran-
detergent, is sensitive to RNase, and requires all scriptase and that on the known DNA polymerases
four deoxyribonudeoside triphosphates, divalent of mammalian cells.
cation (Mg" or Mn"), DTT, and monovalent Viral replication and virus-induced transfor-
cations for optimum activity. The enzyme has not mation can arbitrarily be divided into four steps:
been purified yet, but it is capable of using formation of the provirus, integration of the
(dT)oligo (rA), with high efficiency and provirus, expression of the viral genome, and
(dG)oligo (rC), with less efficiency. (dT)oligo assembly and release of virus particles. As
(dA), is not copied. Product analysis of the expected, many studies have shown that reverse
endogenous reactions from intracisternal type-A transcriptase is required for viral transformation in
326 CRC Critical Reviews in Biochemistry
 tissue culture cells and animals’ 2 3 7-3 since ’ of the compounds are added to the purified
it is required for provirus synthesis. After inte- enzyme assay system. The Ki value of class C com-
gration of the proviral genome into host DNA, the pounds in a purified enzyme system in the absence
maintenance and transcription of the proviral of nonionic detergent is about A t 2 ’ ’ The
DNA does not require reverse transcriptase. There- class C compounds have been shown to bind to
fore, inhibitors of reverse transcriptase after reverse transcriptase both by direct binding
integration do not have any effect. Nevertheless, ’
studies’
33 ’’
” and by kinetic s t ~ d i e s . ~ Both
prevention of synthesis of proviral DNA theo- tight and loose binding have been described.2 I3 ’
retically can be useful if additional retransfor- Two to 14 molecules (with an average of 7 to 8) of
mation events occur.342 A specific inhibitor of inhibitor are bound to 1 molecule of reverse
reverse transcriptase might also be a useful tool t o transcriptase.218J52 The binding is reversible by
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determine if reverse transcription is involved in the addition of nonionic detergent’ ’ that traps
embryogenesis and cytodifferentiation. Finally, a the compounds in r n i ~ e l l e s . ~ ’The~ enzyme
specific reverse transcriptase inhibitor would be activity is completely restored following dissoci-
helpful in studies directed at reverse transcriptase. ation from the compound by nonionic detergent.
It has been shown that the inhibitor acts at a step
B. Classification before initiation of DNA synthesis. Therefore,
Compounds that inhibit reverse transcriptase conditions which facilitate initiation will abolish
can be classified according to their mode of action the inhibitory activity. Examples of this are the
(see Reference 343). The compounds are divided formation of an enzyme template complex, an
into six groups: enzyme-substrate complex or an initiation
complex.2
1. Enzyme-binding agents - These include Unfortunately, all class C compounds are also
ansamycins (rifamycin SV derivatives3 334 4-34 potent to moderate inhibitors of cellular DNA
and s t r e p t o ~ a r i c i n s , 733~4~8 ) calcium e l e n ~ l a t e : ~ ~ polymerases’49345 and to a lesser degree, RNA
’’
For personal use only.

alkaloid extract of Narcissus tazerta L.,3 ’O Pyran polymerase.2 9’ ” Riva et al. questioned the
’
c ~ p o l y m e r , ~ and poly(2-O-methyluridylate).’ specificity of class C compounds based on the
None of these compounds is highly specific for observation that class C compounds also inhibit
reverse transcriptase because they also inhibit some other unrelated enzymes such as glutamate
other DNA polymerases and RNA polymerase oxaloacetate transaminase, glutamate-pyruvate-
to some degree. ’
transaminase, and alkaline pho~phatase.~ Barlati
Rifamycin SV derivatives are probably the best further argued that the inhibitory activity of class
studied inhibitors of reverse t r a n ~ c r i p t a s e . ~ ~ C compounds is due to an exertion of some non-
Following the initial observation of inhibition of specific hydrophobic force on the enzyme^.^
reverse transcriptase by some rifamycin SV deriva- However, Gerard et al.356 reported that the
tives by Gurgo et al.,344 more than 200 deriva- inhibitory activity of class C compounds on reverse
tives were ~ c r e e n e d . ~ This ~ ’ >work
~ ~ ~was based transcriptase was specific since they were unable to
on an assumption that by modifying some side reproduce the observation made by Riva et al.3 5 4
chains of ansamycin, some compounds specific for This discrepancy was probably due to the fact that
reverse transcriptase, (analogous to the specificity the concentration of the compounds used by Riva
of rifamycin to E. coli RNA polymerase) would et a1.354 was much higher than that used by
be discovered, but this hypothetical compound Gerard et aL3’ ti
was not identified. The rifamycin SV derivatives Although a highly specific inhibitor of rifa-
are grouped into three classes according to potency mycin derivatives has not been found yet, many
of inhibition of reverse transcriptase.’ In a crude class C compounds are available.’ y3 334ti y3 ’’
virus lysate system, at a concentration of 100 The structure of these compounds and the
pg/ml, class A compounds are weak inhibitors mechanisms of their action may provide useful
(inhibit less than 25% of reverse transcriptase information for synthesizing additional derivatives
activity); class B compounds are moderate that might be used in developing a specific
inhibitors (inhibit between 25 to 90% of enzyme inhibitor. It appears that both the size of the side
activity); and class C are relatively potent inhibitors chain and the hydrophobicity are important.
(inhibit more than 90% of the enzyme activity). Thompson et al. recently have synthesized more
A similar pattern is obtained when 5 to 10 pg/ml ’’
derivative^.^ One of them, Rifazone 8*, was
December 1975 327
 shown to preferentially inhibit the growth of analogues which specifically bind t o reverse tran-
virus-transformed chick cells in culture.’ 2 4 scriptase.
Furthermore, the cytotoxicity is low at the 4. Template-binding agents - This group
effective concentration. i n c l u d e s a c t i n o m y c i n D , 1 7 8 9 3 7 1 chroma-
mycin? ’ ’
pannomycin? 937 2 adriamycin,’ 7 1 ’
2. Substrate analogues - This includes ara- 7’-’ 75 c i n e r ~ b i n , d~ i~~~t a m y c i n , ’ ~ ~ > ~ ~ ~
’
CTP,’ ,36 2’, 3’-dideoxythymidine triphos- ethidium b r ~ m i d e , ~ 71937433783379 profla-
phate (ddTTP),’ and cordycepin triphosphate vine: 7 4 tilorone and its cogeners fluroanthene
(Gallo and Wu, unpublished data). All these derivative^,^ y3 ’ s4 acridine Orange, congo
inhibitors are nonspecific as predicted from their red, histone, and p r ~ t a m i n e . ’ ~ ’ Many of the
Critical Reviews in Biochemistry and Molecular Biology Downloaded from informahealthcare.com by 198.189.249.60 on 11/02/14

mode of action. commonly used antibiotics belong to this group.

They inhibit DNA synthesis by binding to some
3. Primer-template analogues - This group specific bases. For example, actinomycin D binds
of compounds exerts an inhibitor activity by to guanosine bases while daunomycin, distamycin,
either tightly binding to reverse transcriptase or and tilorone are adenine-thymidine base-specific.
competing with primer-template for its binding 5. Divalent cation-binding agents - By
site. Since the binding of primer-template to definition, any chelating agent which removes
nucleic acid polymerase generally is not specific, it divalent cations required for DNA synthesis could
is rather difficult to obtain a specific inhibitor of be an inhibitor of DNA polymerase. 0-phenan-
this group. Although some primer-templates such t h r 0 1 i n e ~ ~and ~ thiosemicarbazones160 are
as HMW RNA are specific for viral reverse tran- examples of these compounds. Apparently, they
scriptase activity, it is not known whether the are not specific inhibitors of any polymerase
binding is also specific. Compounds which fall into including reverse transcriptase.
this group include thymidylate derivatives? ti ’ 6. Agents of unknown mode of action -
polyribonucleotides: 2-’ ’ modified polyribo- Streptonigrin is a potent inhibitor of focus forma-
For personal use only.

nucleotides such as thiolated polycytidylate and tion but a relatively poor inhibitor of reverse
natural RNA? ’,’ and 2’-O-alkylated p l y - transcriptase.’ Bleomycin inhibits the DNA-
adenylic acids.’ 7 0 Among four homopolymers dependent DNA synthesis reaction of reverse
tested, the order of their potency was as follows: tran~criptase.’~’ It inhibits RNAdependent DNA
( U ) , > ~ G ) , > ~ A ) , ~ C ) , .’ Low concentra- synthesis only at a high concentration. It is a
tions of (U), have a higher affinity to viral reverse noncompetitive inhibitor with the DNA primer-
transcriptase than other cellular DNA polymerases template. Other compounds such as 2-oxopro-
except DNA polymerase y.j6’ However, high panal: 7 2 heparin: ’ and silicotungstate’ 8 4 are
concentrations of (U), are non~pecific.’~’ The also inhibitors of reverse transcriptase. Heparin
size of (U), affects its potency. The minimum seems to be specific for the RNA template since it
chain length is 200 n u ~ l e o t i d e s . ’ ~Thiolation
~ of does not inhibit RNA and DNA polymerases when
(C), or some other natural template (even yeast S DNA is used as template.
RNA) improves the potency and the specificity of
the polymer as a reverse transcriptase inhibi- V. BIOLOGICAL ROLES OF
tor.’ ’ 9’ Among many partially thiolated REVERSE TRANSCRIPTASE
polycytidylates, Chandra and Bardos observed that
one of them, MPC I11 (2-6% cytosine bases are A. Reverse Transcriptase and Proviral Synthesis
thiolated), is relatively a potent inhibitor.’ The The only well-defined biological role of viral
other type of modification obtained by substitu- reverse transcriptase is its requirement for success-
tion in four and five positions of the base ring and ful viral infection and transformation, i.e., for
two position on the sugar,such as poly(flurodeoxy- provirus synthesis. Two types of evidence support
uridylic acid), poly(chlorodeoxyuridylic acid), this statement. One is the evidence based on
poly(chlorodeoxycytidy1ic acid), and poly(bromo- studies using reverse transcriptase inhibitors. There
uridylic acid), has been found to significantly alter is a direct correlation between the inhibition of
the inhibitory activity.’ With increasing in- viral transcriptase activity and viral transformation
formation on the nature of the primer in various in c ~ l t u r e ’ ’ ~and of virus induced leukemia in
type-C virus systems, it may be possible to develop anirnal~’~’ by various classes of rifamycin SV

328 CRC Critical Reviews in Biochemistry

 derivatives. The other is evidence based on genetic organisms (proliferating or nonproliferating) con-
studies. Hanafusa and Hanafusa isolated a mutant tain authentic reverse transcriptase, although there
’’
of RSV called RSVNO) which is d e f e ~ t i v e . ~ are some observations which are suggestive.’ 03,
This mutant has been shown to lack reverse ’ There is some evidence indicating that reverse
transcriptase both by b i ~ c h e m i c a l ” ~ and transcriptase is present in embryonic tissues. As
immunological3 * criteria. Recently, Mason et described in Section III.B.3, Kang and Temin
aL419 and Verma et al.263 demonstrated that found RNase-sensitive DNA synthesizing activity
reverse t r a n s c r i p t a s e purified from two in chicken embryonic cells.’ O 4 The endogenous
temperature-sensitive mutants of RSV that are DNA product is found in a hybrid form” and ’
defective in transf~rmation’~’ were also the isolated DNA product is able to back hybridize
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temperature-sensitive for DNA synthesis in vitro. to RNA isolated from the cytoplasmic pellet
Moreover, intracellular viral DNA was not syn- fraction.’ O 4 33 ’’
Mayer et al. isolated a reverse
thesized when duck embryo cells were infected by transcriptase activity from rhesus monkey
these two mutants at a nonpermissive temperature placentaembryo.’ This reverse transcriptase
(41°C).42 y4 ’’ Similarly, Tronick et al. reported activity is immunologically related to viral reverse
that enzyme purified from a temperature-sensitive transcriptase of the M7 virus (isolated from normal
mutant of R-MuLV ( t ~ 2 9 ) , ~defective~’ in an baboon placenta and now known t o be an endo-
early function of virus replication and sarcoma genous virus), but not to those of known
virus helper function, was also temperature- oncogenic primate viruses. In addition, type-C-like
sensitive for DNA synthesis in ~ i t r o . ~These’~ particles have been visualized in human,’
findings prove that the reverse transcriptase ’
c h i m p a n ~ e e , ~ baboon,’ ’ and rhesus
molecule is required for the synthesis of proviral monkey’ ’ placentas, and virus-like particles have
DNA in vivo and therefore, for transformation been seen in baboon’ ’ and mouse’ 9 4 ,’ pre-
and infectivity by typeC virus. implantation embryos. These findings are in keep-
ing with the idea that the expression of fetal
For personal use only.

B. Reverse Transcriptase from Normal Tissues antigens and “virogenes” are related. Furthermore,
The discovery of transcriptase is basically a another viral component, gp70, is found closely
fulfillment of the prediction made in Temin’s linked to lymphoid cell development and differen-
’
provirus hypothesis.2 An important by-product tiation which is mediated through the GI,
of this discovery is the revelation of a new pattern marker! ’’ ’ ,4

of genetic information transfer, RNA to DNA,

which in turn invites new speculations. It is C. Classification of Reverse Transcriptase
possible that the reverse flow of viral genetic We like to view intracellular reverse transcrip-
information is only one example of a mechanism tase as two different types, intrinsic and extrinsic.
more commonly employed. This mechanism could The function of extrinsic reverse transcriptase, as
involve repeated cycles of transcription, reverse described above, is clear but that of intrinsic
transcription, and recombination that could lead reverse transcriptase is not. Concerning reverse
to somatic mutation and the generation of new transcriptase, the questions that appear to us to be
information during the course of somatic cell of the greatest interest for the future are related to
development and differentiation. In fact, these the intrinsic enzyme. (1) Do all normal cells
are the basis of Temin’s protovirus hypothesis contain the genome for reverse transcriptase? (2)
for the generation of viral gene^,'"^''^ When is it expressed and what is the function of
neoplasia’ 8 6 and normal development.’ “ “intrinsic” reverse transcriptase? (3) Are there
Similarly, reverse transcriptase has also been pro- functional relationships between intrinsic and
posed to play some role in ribosomal gene amplifi- extrinsic reverse transcriptase? What the authors
’
cation3 ,3 ” and in the genesis of various clones present below is hypothetical and is designed to
of precursors of antibody-producing cells.’ ’ conclude this review by stimulating further
Experiments to support these proposals, however, studies.
are not yet convincing.
These concepts must be supported first by clear I . Intrinsic Reverse Transcriptase for Development
evidence that normal cells contain authentic and Differentiation
reverse transcriptase. At present, there is no The meaning of development here is the for-
definite evidence that adult tissues of higher mation of stem cells for various functions from the

December 1975 329

 Development Differantiation

FIGURE 1. Diagrammatic representation of sequence of development and differentiation.

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TABLE 11
Interrelationship Between Control of Proliferation and Presence of Reverse TranscriptPse, Fetal Antigen, and Viral Antigen

Reverse Transcriptase

Fetal Viral
Control of proliferation Intrinsic Extrinsic antigen antigen

Developing tissues Relaxed Present* N.R.** Present Present

Differentiating tissues: early Stringent Present N.R. N.R. N.R.
late ? N.R. N.R. N.R.
Malignant tissue Aberrant, generally relaxed ? Present Present Present

*This does not mean adequate data have been obtained. The terms refer only to what the hypothesis predicts.
**N.R.: Not required.
For personal use only.

fertilized egg, and differentiation, as the develop- one would predict nonidentity of the genetic
ment of mature functional cells from stem cells information among various types of stem cells. In
(Figure 1). The fertilized egg, by definition, is fact, there is no evidence that all stem cells contain
pjuripotent and unable to renew itself. Stem cells identical genetic information as do germ cells or
are multipotent. They can give rise t o more than fertilized eggs. The studies of Gurdon and
one kind of differentiated descendant cell. All Woodland396 showed that at least some must have
physiological functions are carried out by the the complete information contained in the
mature cells, such as granulocytes, erythrocytes, fertilized egg while the studies of Briggs and King
sperm, muscle, bone, etc., but they are not able to showed that nuclear activity changed at or near
divide and therefore stem cells must be able to the onset of gastrulation.’ ’
Fetal antigens also
renew themselves to provide a continuous supply appear at this state of development. The role of
of mature cells. The process of differentiation can these antigens is not known. They might have
be further divided into early and late stages. Early some regulatory function or they might be just a
differentiation involves the formation of unipotent by-product of gene expression. In some mouse
progenitor cells from stem cells and the late embryos, type-C particles and viral antigens are
differentiation includes all the processes thereafter frequently detected.’ ’ 9’
9’ ’
However, these
(see Figure 1). a r e probably extrinsic reverse transcriptase
During the course of development, the control resulting from congenital infection. It is, of course,
of proliferation is relatively relaxed (Table 11). not known whether they Serve any function in
Relaxed proliferation is probably required for n o r m a l development. Upon completion of
maximum development. This is the period during development of a given system, differentiation of
which the authors believe intrinsic reverse stem cells occurs corresponding to physiological
transcriptase will be used, and in fact, where it has demands, and therefore, more stringent control is
been detected. The exact function, if any, of this required. This type of stringent control is not
reverse transcriptase is, of course, not known. necessary for the process of development due to
During the development of various types of stem the simple environment. In early differentiation
cells, they may require a gene modification and/or when progenitor cells are formed, intrinsic reverse
amplification. Intrinsic reverse transcriptase might transcriptase might play a role in modifying
be the catalyst for this purpose. If this is the case, genetic components for differentiation, such as
330 CRC Critical Reviews in Biochemistry
 generating various clones of progenitors of anti- differentiation. However, the reversion of tumor
body producing cells. To support this hypothesis, cells to normal cells is not impossible if the genetic
definite proof of the presence of intrinsic reverse lesion is not severe. An example of such conver-
transcriptase in normal proliferating cells is sion is the formation of mature granulocytes from
required. There is no evidence of fetal antigens at leukemic blast cells in semi-soft agar medium in
this stage. At the later stages of differentiation, the the presence of a protein factor, termed CFS404
pattern of ceIlular proliferation is more confined. or in liquid suspension.4oSy416To explain the
This might involve some gene reduction, such as appearance of fetal antigens in some tumor tissues,
exonucleation by erythrocytes and extrusion of one might assume that the gene affecting the
DNA by some lymphocytes, but it would appear regulation of cellular proliferation linked to the
virogene is also linked to some fetal antigen
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unlikely that gene modification occurs at this

stage. Intrinsic reverse transcriptase and fetal genomes (either regulatory or structural). A distur-
antigens are, of course, not required at this stage. bance of proliferation will then lead to an
expression of fetal antigen genes. For example, the
2 . Extrinsic Reverse Transcriptase and S2 antigen found in some human sarcomas,"o6
Carcinogenesis car c i n o e m b r y o n ic antigen:'' alpha feto-
Extrinsic reverse transcriptase is introduced protein?'* and many other placental and fetal
into cells upon infection by an oncornavirus. The ''
protein^.^ O 9 94 are found either in the blood or
source of extrinsic reverse transcriptase can be by in the tumor tissues of cancer patients. If these
infection from without or can be produced from findings are related to a linkage of fetal antigen
within. Its function is to synthesize proviral DNA. genes and proliferation genes, it will imply that the
E x t r i n s i c reverse transcriptase is normally expression of fetal antigens in cancer is a by-
associated with oncogenic RNA viruses. It may product rather than a result of the systematic
have evolved from intrinsic reverse transcriptase. phasing process of embryonic antigens as suggested
Extrinsic reverse transcriptase might cause a distur- by Coggin and A n d e r ~ o n . ~ 'In fact, not all of the
For personal use only.

bance in cellular proliferation in two possible fetal antigens are found in all cancers and some are
ways. First, extrinsic reverse transcriptases might even found in some patients not known to have
directly cause aberrant gene modification or cancer.406 94 1 2 94 I 3
inappropriate gene amplification. In fact, Loeb
and his colleagues have repeatedly observed that VI. CONCLUSION
viral reverse transcriptase, and probably some
human leukemic DNA polymerase as well, have a A wealth of information is now available on the
higher rate of miscopying homopolymers in biochemical, biophysical, and immunological
~ i t r o . ~ ~ Th ~ ,e~rate
' ' of infidelity is even higher properties of various reverse transcriptases. The
if heteropolymers or natural primer-templates are enzyme has been of great help for the identifica-
used. Second, the provirus might integrate into tion and quantitation of virus, for preparing
some specific location such as a "hot a molecular probes, as an indication of virus in
Tr gene,402 an oncogene,38' or some prolifera- tissues not known to be producing virus; and, of
ting regulatory genes, resulting in a direct or course, for providing major information for our
indirect effect on the stringent control of pro- understanding of the replication of these viruses.
liferation through derepression or induction of The origin and the role of the enzyme is clear in
some relevant genes. In fact, it has been observed virus-infected cells. What is unclear and potentially
that cellular transformation induced by viral more important relates to the possibility that
infection may occur prior to cell p r ~ l i f e r a t i o n . ~ ' ~ reverse transcriptase, distinct from that of
It is generally observed that transformed cells have infecting viruses, may have a role in development
an abnormal mode of cellular proliferation. This and differentiation. The authors of this review call
abnormality probably is induced by some gene this "intrinsic" reverse transcriptase, and they
product related to transformation. This is an think that it is involved in gene modification
example of an indirect effect of extrinsic reverse and/or gene amplification during development.
transcriptase. The authors propose that extrinsic reverse tran-
Once relaxed control of proliferation occur's in scriptase, besides being able to synthesize provirus,
adult tissues, it is difficult to convert back to may cause abnormal cell proliferation either by
stringent control, since the control element has affecting gene modification or gene amplification,
already been modified during the course of or by integrating the provirus into the host
December 1975 331
 chromosomes at some specific location. Support Sarngadharan and R. G. Smith for useful discus-
for some of these concepts will require, among sions, Cillian Wu and Dr. Sarngadharan for
many other studies, definite proof of the existence editorial assistance, and Vicki McConnell and
of intrinsic reverse transcriptase in a variety of JoAnn Fleishman for secretarial assistance. No
cells and especially its identification in normal official support or endorsement from the Depart-
adult cells. ment of Health, Education, and Welfare, National
Institutes of Health, or National Cancer Institute
ACKNOWLEDGMENTS
was obtained or utilized by Dr. Robert Gallo for
The authors wish to thank Drs. M. the preparation of this review.
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GLOSSARY

AMV - Avian myeloblastosis virus. M-MuLV - Moloney strain of murine

ccc - An isolate of feline leukemia virus.
endogenous virus. M-MuSV - Moloney strain of murine
csv - Chicken syncytial virus. sarcoma virus.
FeLV - Feline leukemia virus. R-MuLV - Rauscher strain of MuLV.
FeSV - Feline sarcoma virus. C-MuV - Caroli murine virus.
C-FeLV - Gardner strain of FeLV. PK (1 5) virus - Endogenous type-C virus
G-FeSV - Gardner strain of FeSV. isolated from porcine cell
R-FeLV - Richard strain of FeLV. line PK. '
For personal use only.

R-FeSV - Richard strain of FeSV. RaLV - Rat leukemia virus.

T-FeLV - Thelian FeLV. RAV - Rous associated virus.
GaLV (SEATO) - Gibbon ape lymphosar-
RAV-1 - Strain no. 1 of Rous
coma virus isolated from
associated virus.
SEATO (Thailand) gibbon
RAV-2 - Strain no. 2 of Rous
ape colony.
associated virus.
GBr-1,
RD-114 - Feline endogenous virus
CBr-2,
induced by and grown in
and human rhabdomyosar-
GBr-3 GaLV-like virus isolated coma (RD) cells.
from brain tissues of REV - Reticuloendotheliosis vi-
three gibbon apes obtained rus.
from Southeast Asia. REV-T - Reticuloendotheliosis
HaLV - Hamster leukemia virus. (strain T).
HaSV - Hamster sarcoma virus. RSV - Rous sarcoma virus.
M-7 - Baboon endogenous virus B77 - B77 strain of RSV.
isolated from placenta. B-RSV - Bryan high titer strain
MMTV - Mouse mammary tumor of RSV.
virus. RSV (RAV - 0) - RSV with pseudotype
MPMV - Mason-Pfuer monkey Rous associated virus4
virus. (RAV-0).
H-MuSV - Harvey strain of murine PR-RSV - Prague strain of RSV.
sarcoma virus. Pr-RSV/A - PR-RSV subgroup A.
F-MuLV - Friend strain of murine PR-RSV/B - PR-RSV subgroup B.
leukemia virus. PR-RSV/C - PR-RSV with subgroup C.
K-MuLV - Kirsten strain of MuLV. SR-RSV - Schmidt Ruppin strain of
K-MuSV - Kirsten strain of MuSV. RSV.

332 CRC Critical Reviews in Biochemistry

 SR-RSV/A - SR-RSV subgroup A. LMW - Low molecular weight.
SiSV SiLV - Simian sarcoma leukemia NEM - N-ethylmaleimide.
virus. DMSO - Dirnethylsulfoxide.
TD SNV - Trager duck spleen neuro- DTT - Dithiothreitol.
sis virus. I@ - Immunoglobulin G .
SV40 - Simian virus 40. NC-37 cells - A normal human lympho-
HMW - High molecular weight. blastoid culture cell.
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December 1975 333

 22. Temin, H. M., Homology between RNA from Rous sarcoma virus and DNA from Rous sarcoma virus infected cells,
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