REVIEWS

The cell biology of rabies virus:

using stealth to reach the brain
Matthias J. Schnell*, James P. McGettigan*||, Christoph Wirblich*|| and
Amy Papaneri*
Abstract | Rabies virus, the prototypical neurotropic virus, causes one of the most lethal
zoonotic diseases. According to official estimates, over 55,000 people die of the disease
annually, but this is probably a severe underestimation. A combination of virulence factors
enables the virus to enter neurons at peripheral sites and travel through the spinal cord to
the brain of the infected host, where it often induces aggression that facilitates the transfer
of the virus to a new host. This Review summarizes the current knowledge of the replication
cycle of rabies virus and virus host cell interactions, both of which are fundamental elements
in our quest to understand the life cycle of rabies virus and the pathogenesis of rabies.

Rabies is a devastating and important, albeit neglected, disease10. Therefore, new approaches for therapy and
infectious disease that has been feared by mankind for cheaper, more effective vaccines are urgently needed.
more than 4,000 years1. Although rabies was not recog- A better understanding of the biology of the virus
nized to be the result of an infection when it was first might also help to develop rabies virus as a tool for
described, it was well known that a bite from a mad neuronal labelling and neurotracer studies1114 and will
dog would most likely result in the death of the bitten provide insight that will facilitate the development of
individual, and laws regulated the consequences for the treatments for other diseases of the central nervous
dogs owner 2. In 1802, George Zinke3 showed that rabies system (CNS).
could be transmitted from the saliva of a rabid dog to In the past, the study of rabies virus was hampered
a healthy dog and cause disease. Pierre-Victor Galtier 4 by the lack of genetic manipulation; therefore, research
first demonstrated the transfer of rabies from one ani- studies were based on the use of different rabies virus
mal species to another, and his studies were continued strains, which were often genetically diverse. Although
and extended by Louis Pasteur 2, who established that these studies enhanced our understanding of the virus,
the virus is harboured in the brain. In 1885, Pasteur the reverse genetics technique established in 1994 to
developed the first rabies virus vaccine from the spi- directly manipulate the rabies virus genome has opened
nal cord of rabbits with rabies5. This meant that rabies, new research areas for rabies virus molecular biology
which is almost always fatal after the onset of disease, and pathology 15. The ability to introduce precise muta-
was no longer an automatic death sentence for those tions and to exchange genes from vaccine and patho-
exposed6,7. genic strains has allowed more detailed analysis of the
Despite the great progress that has been made in virushost cell interaction. In this Review, we describe
Department of Microbiology
and Immunology*and
the development of rabies virus vaccines and the con- our current knowledge of the cell biology of rabies virus,
Jefferson Vaccine Center, trol of rabies (for a review, see ref. 8) (BOX 1, fIG. 1), the including the steps of the viral life cycle and virushost
Jefferson Medical College, annual number of deaths caused by rabies virus world- cell interactions.
Thomas Jefferson University, wide is estimated to be between 40,000 and 70,000 (see
Philadelphia, Pennsylvania
WHO Fact Sheet No. 99). However, a recent study in Genomic organization and replication
19107, USA.
||
These authors contributed
Tanzania indicated that the number of deaths caused Rabies virus belongs to the genus lyssavirus (from the
equally to this work. by rabies may be up to 100 times higher than officially Greek word for frenzy) in the Rhabdoviridae family.
Correspondence to M.J.S. reported 9, and it has been suggested that only 3% Rabies virus is divided into two phylogroups with a total
e-mail: Matthias.schnell@ of human cases of rabies virus are recorded by cen- of 11 genotypes16,17. Genotype 1, which contains the so-
jefferson.edu
doi:10.1038/nrmicro2260
tral health authorities1. As 40% of those infected are called classical rabies virus (see BOX 2 for definitions) is
Published online children, the years of life lost by this infection makes the most prevalent and is responsible for most animal
30 November 2009 rabies the seventh most important global infectious and human infections and deaths16.

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REVIEWS

Box 1 | Rabies symptoms and treatment likely enters a motor neuron at the neuromuscular
junction. In either case, rabies virus particles are sub-
Clinical manifestation of rabies in humans has two forms7: the furious (classical) sequently transported in a retrograde direction through
form (80% of infections), and the numb (non-classical or paralytic) form (20% of the axon of the infected neuron11. Once the virus parti-
infections)126. The furious form of rabies is characterized by hydrophobia: terror
cles reach the cell body of the neuron, the second phase
and excitation with spasm of inspiratory muscles, larynx and pharynx precipitated
commences, which involves the production of the vir-
by attempts to drink (ref. 7), and episodes of hallucinations and excitement are
common. Animals often present with extreme aggression and randomly attack ion components (transcription, replication and protein
objects, other animals or humans127. These behavioural changes occur synthesis). The last phase of the life cycle comprises the
simultaneously with the shedding of large amounts of rabies virus in the saliva, assembly of the viral components, their transport to the
which facilitates the spread of the virus to a new host. The numb form of rabies site of budding and the release of mature rabies virus
is characterized by weakness and flaccid paralysis, which sometimes causes particles, which can then start a new round of infection.
misdiagnosis at the onset of this clinical form of rabies126. In both cases, survival every step of this process is highly regulated and only
after the onset of symptoms is rarely more than 7 days7. partly understood (fIG. 3).
Human rabies virus infections are controlled by limiting the infection in animals and
by post-exposure prophylaxis (PEP) of humans after contact with a potentially infected
The rabies virus receptor still a mystery? The first
animal. Current PEP in the United States for previously non-immunized individuals
step in the rabies virus life cycle is the attachment to the
consists of one dose with rabies immunoglobulin in conjunction with five doses of
immunization with inactivated rabies virus vaccines128. This regimen is effective, but its host cell. This step requires the rabies virus glycopro-
usefulness in developing countries, where rabies is endemic, is hampered by the high tein. Indeed, rabies viruses with a deletion in the gene
cost and lack of compliance. The WHO has approved alternative vaccine regimens in encoding the glycoprotein that are trans-complemented
developing countries to reduce the cost of vaccination. Approaches that do not rely on with the glycoprotein infect cells efficiently but can-
the use of inactivated vaccines, including DNA vaccines129134 and viral vectors135140, are not spread from the infected cell in vivo23 or in vitro 24,
also being investigated. Although pre-exposure vaccination has been historically indicating that the glycoprotein is required for the
reserved for populations at risk of infection, such as veterinarians and laboratory spread of the virus. However, it is less clear which
workers, efforts are underway to promote and determine the efficacy, safety and cost host cell molecule (or molecules) interacts with
for a broader application of rabies virus vaccines in pre-exposure settings, especially
the rabies virus glycoprotein and mediates its entry
children in high risk areas141143.
into cells. Nicotinic acetylcholine receptor (nAchR)
was the first identified potential receptor for rabies
Rabies virus is the prototypical neurotropic virus and virus25. Because nAchRs are located at the postsynaptic
has a small, negative-stranded RNA genome of about muscle membrane and not at the presynaptic nerve
12 kb, which encodes five proteins18,19 (fIGS 2,3). Similarly membrane26, it is unlikely that this receptor is used
to all negative-stranded RNA viruses, the RNA genome for the initial entry into motor neurons. Instead, it is
is tightly encapsidated into the nucleoprotein. Only the possible that nAchR enriches rabies virus at the neuro-
encapsidated RNA, termed the ribonucleoprotein (RNP), muscular junction to enable more efficient infection
is a functional template for transcription and replication. of the connected motor neurons26 (fIG. 4). moreover, as
The viral capsid also contains the viral polymerase com- rabies virus might initially replicate in striated muscle
plex (PC), which consists of the polymerase (known as cells, nAchRs could be used to infect muscle cells27,28.
L, for large protein) and the phosphoprotein, the non- Other potential receptors for rabies virus include
catalytic subunit of the polymerase complex. The capsid is neuronal cell adhesion molecule (NCAm; also known
surrounded by the host cell-derived membrane that as NCAm1)29 and low-affinity nerve growth factor
interacts with two viral proteins, the matrix protein receptor (p75NTR; also known as BeX3 and NGFR)30.
and the glycoprotein. On a structural level, the matrix It has been shown that virus-resistant cell lines are
protein functions as a bridge between the rabies virus permissive to rabies virus infection after NCAm
RNP and the virion membrane. The matrix pro- expression and that NCAm-specific antibodies and
tein interacts with and condenses the RNP, yield- NCAm ligand treatment reduce rabies virus infec-
ing the typical helical form of the RNP found within tion29. However, mice in which the gene that encodes
the virions. It also interacts with the cytoplasmic NCAm was deleted were still susceptible to infection
domain of the glycoprotein, which forms a trimer 20, with the virus, although the disease was delayed. This
and has a role not only in virus entry and membrane indicates that although the receptor might not be essen-
fusion, but also in virus release21. tial for infection, it has a role in the infection process29.
The function of p75NTR in rabies virus entry is less
The life cycle of rabies virus clear. Similarly to NCAm, expression of p75NTR ena-
As for most viruses, the rabies virus life cycle can be bles non-permissive cells to be infected with a rabies
divided into three phases (fIGS 3,4). The first phase virus field isolate, but there is no difference in disease
Trans-complemented
includes binding to the host cell receptor (or receptors), progression between p75NTR-deficient and wild-type
Complemented by expression
of missing components in the entry into the host cell by endocytosis followed by fusion mice31. moreover, only 25% of the primary dorsal root
host cell. of the viral and endosomal membrane and release of the ganglions that express p75NTR were infected by rabies
viral genome into the cytoplasm (known as uncoating). virus ex vivo, indicating that p75NTR might only func-
Postsynaptic Although a recent study using bat-associated rabies tion in combination with another cell surface mole-
Positioned after the synapse.
virus has shown that the virus spreads in the blood and cule31. Carbohydrates32,33, gangliosides32 and lipids34
Presynaptic invades the CNS at the neurovascular junction of the have also been suggested to be rabies virus entry receptors,
Positioned before the synapse. hypothalamus22, classical wild-type rabies virus most but their functions are not well established.

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a b

No data available 0
Rabies present 11,000
1011,000
Rabies absent
1,00110,000
Only bat rabies
>10,000
present

Figure 1 | rabies worldwide. a | The worldwide distribution of rabies virus. The distribution of classical or terrestrial
Nature Reviews | Microbiology
rabies virus (genotype 1 (GT1)) in 2007 is shown in orange. The distribution of rabies virus and other members of the
lyssavirus genus is shown in yellow. The only known lyssavirus-free country is New Zealand (green). b | Number of
human deaths from rabies in 2004. The map indicates the areas in South-East Asia most affected by human deaths
caused by rabies for which reliable data exist.

None of the multiple potential receptors identi- fusing at low pH levels is that the inhibition of fusion
fied seems to be essential in vitro. The finding that a is reversible40. It remains unclear whether uncoating in
single point mutation in the rabies virus glycoprotein primary neurons occurs immediately after internaliza-
can render rabies virus non-neurotropic and reduce its tion or only after transport through the axon in the cell
uptake speed35 can only be explained by the use of two body; this is discussed in more detail below.
different receptors. Differences in speed of uptake and
spread in tissue culture of bat-derived rabies virus and Intracellular rabies virus transport. Because the entry
a rabies virus vaccine strain also suggest that different site of rabies virus in axons does not provide the bio-
receptors may be used36. It is possible that certain recep- chemical environment required for protein synthesis41,
tors require an additional molecule (a co-receptor), as rabies virus needs to reach the neuronal cell body for
suggested for p75NTR31. Distinguishing the receptor replication and transcription. Two different mechanisms
mechanism is even more complicated for in vivo systems have been proposed for the transport of rabies virus
because it is unclear whether the receptor (or receptors) through the axon to the cell body: transport of either the
used for the initial infection is the same as the one used rabies virus capsid alone or transport of the whole virion
for viral spread35. Obviously, more research is needed (fIG. 4). The transport of the rabies virus capsid would
to understand the function of rabies virus receptors for require uncoating after entry and a specific interaction
viral entry, spread and pathogenicity. of the RNP with the cellular transport machinery. Two
research groups42,43 identified an interaction between
Internalization. Rabies virus particles can be found in rabies virus phosphoprotein and the dynein light chain 8
clathrin-coated pits, in uncoated vesicles and, at later (LC8), which led them to propose that the virus uses
time points after infection, in lysosomes32. The exact the cytoplasmic dynein motor complex for intracellular
mechanism by which rabies virus is internalized into transport. However, deletion of the LC8-binding site in
these compartments remains unclear. Fusion of the virus the phosphoprotein does not affect viral transport from
with these vesicles requires a change in the pH inside the a peripheral site to the CNS44,45, and LC8 directly or indi-
vesicle. This has been shown by ammonium chloride- rectly affects primary transcription of rabies virus but
and chloroquine-induced inhibition of membrane not its transport 45.
fusion and release of the rabies virus capsid32 and by the A recent study used a capsid- and envelope-labelled
loss of pH dependence when the virus contains a foreign rabies virus and showed that the whole virion is trans-
glycoprotein, such as HIV-1 gp160, instead of the rabies ported to the cell body 46 in an endosomal vesicle (fIG. 4),
virus glycoprotein37,38. Thus, the rabies virus glycopro- although the exact mechanism by which this occurs
tein is solely responsible for both attachment and pH- remains unclear. Transport depends on the rabies virus
dependent membrane fusion. The crystal structure of glycoprotein, as retroviral vectors trans-complemented
the glycoprotein of a related rhabdovirus, vesicular sto- with rabies virus glycoprotein are transported in a simi-
matitis virus (VSV), revealed that this protein combines lar manner as rabies virus4750. However, as the rabies
structural features of both class I and class II fusion pro- virus glycoprotein is inside the vesicle, it should not be
teins39, and a similar structure has been suggested for the able to interact directly with a specific transporter com-
rabies virus glycoprotein40. A major difference of these plex. Based on these observations, it is speculated (but
rhabdoviral glycoproteins from those of other viruses not shown experimentally) that entry into the vesicle,

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Box 2 | Definitions of rabies virus strains as diagnostic markers for rabies virus infection. Negri
bodies were shown to contain ubiquitylated proteins and
Rabies virus street virus: an isolate from a naturally heat shock protein 70 (HSP70); they are not aggresomes
infected animal, for example a dog or fox. containing misfolded proteins but instead are functional
Fixed rabies virus: a rabies virus that has been passaged structures for viral replication53. Interestingly, the for-
in tissue culture or animals. Fixed rabies virus can be mation of Negri bodies depends on Toll-like receptor 3
pathogenic or non-pathogenic. The term fixed indicates (TLR3)54, which is involved in the spatial arrangement
only that the incubation period and virulence has been
of sites of rabies virus replication.
stabilized.
After the release of the RNP into the host cell cytosol,
Pathogenic rabies virus: a strain that typically causes the condensed and helical form of the RNP needs
rabies after peripheral inoculation.
to switch to a relaxed stage to be synthetically active.
Attenuated rabies virus: a strain with a greatly reduced Direct translation of viral messenger RNA cannot occur
ability to cause rabies after inoculation into an animal.
because the viral genome is negative sense RNA. In addi-
Neurotropic rabies virus: a strain that preferentially tion, the encapsidation of the genome and anti-genome
infects primary neurons or neuronal cell lines. by nucleoprotein18,19,55,56 would interfere with this proc-
Non-neurotropic rabies virus: a strain that infects ess. Crystal structure analysis using ring complexes of
neuronal cells at a level less than or equal to other cell 11 nucleoprotein molecules and single-stranded RNA
types.
revealed that the nucleoprotein consists of two main
domains, which contact nine nucleotides of RNA.
Polymerization between the nucleoprotein protomers
which is mediated by the rabies virus glycoprotein, allows the formation of the RNP57. This structure nicely
determines the direction and provides the driving force explains the protection of the RNA and the stability of
of rabies virus transport. This indicates that the nature of the RNP, but it does not reveal the molecular and struc-
the vesicle formed dictates the transport method that tural basis of the preferential encapsidation of viral RNA
follows. However, this theory is at odds with the finding compared with host cell RNA (see below).
that different receptors are used by different rabies virus The PC, which is found in the rabies virus capsid,
strains, unless entry using different receptors results in synthesizes the initial viral RNAs. Transcription begins
internalization into similar vesicles. at the 3 end of the genomic RNA and results in a short
uncapped and unpolyadenylated leader RNA (leRNA) of
Replication, transcription and protein expression. 5558 bp. This is followed by sequential synthesis of 5
Rabies virus gene expression and genome replication end-capped and polyadenylated mRNAs that encode the
is highly regulated and differs in some respect from viral proteins19,56. The most widely accepted model for
that of other rhabdoviruses, including VSV. Although transcription of rhabdoviruses is the so-called stopstart
it seems that VSV aims to produce as much virus in the model, in which the PC stops transcription at a conserved
least amount of time, rabies virus endeavours to preserve signal sequence, ignores the intergenic region (IGR) of
infected cells for two reasons. First, induction of cyto- 224 nucleotides and restarts at the transcription start
toxicity in the infected neuron would probably prevent signal sequence58 (fIG. 3). Successful reinitiation of tran-
neuronal transport. Second, the production of high lev- scription at each gene junction does not always occur,
els of viral protein, especially glycoprotein, would induce so transcription is attenuated from the 3 to the 5 end
strong humoral immune responses51, which would prob- of the viral genome59. As such, the gene order partly
ably neutralize the virus before it could complete its life determines the level of the respective viral mRNAs. The
cycle. Therefore, rabies virus has evolved regulatory number of nucleotides in the IGR also regulates the tran-
mechanisms that produce viral components at the opti- scription levels of the viral mRNAs59 (fIG. 3). unlike VSV,
mum amounts necessary for efficient viral production in which the IGRs always consist of 2 nucleotides60, the
but low enough not to be recognized by the immune IGRs in rabies virus are 2, 5, 5 and 2429 nucleotides
system or to interfere with the vital functions of its host long 18,19. Switching the IGRs in the rabies virus genome
cells. most studies analysing the interactions of rhabdo- alters the mRNA and protein levels of the downstream
viral proteins with cellular proteins for replication and genes59, indicating that the length of the IGRs has an
transcription have been carried out using VSV and have important role in regulating mRNA expression levels61.
been reviewed previously 52. Although there are still lim- The leRNA also regulates the transcription of rabies
ited data on cellular cofactors of the rabies virus replicase virus genes62. The nucleoprotein binds to the leRNA
Aggresome and transcriptase compared with what is known about about 10-fold stronger than to other rabies virus mRNAs;
An inclusion body in which the VSV proteins52, it is not obvious that the same inter- therefore, it has been proposed that the leRNA contains
proteins accumulate when the
actions exist for rabies virus. The finding that the dynein the encapsidation signal for the nucleoprotein63. It is
cells degradation machinery is
not functioning properly or is LC8 serves as an important factor for primary transcrip- speculated that when sufficient nucleoprotein is avail-
overwhelmed. tion of rabies virus is an example for differences between able, encapsidation of the leRNA results in the synthesis
the two viruses45. of an encapsidated full-length anti-genome rather than
Toll-like receptor Recent research strongly indicates that replication and the production of individual viral mRNAs, switching
A receptor that recognizes
viral, bacterial or fungal
transcription of rabies virus occurs in Negri bodies, which from transcription to replication 64. This hypothesis
material and signals to induce are cytoplasmic inclusion bodies that are typically found is supported by findings that rabies virus tolerates a
an immune response. in rabies virus-infected neurons53 and have been used large range of non-rabies virus RNA sequences in its

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Glycoprotein Matrix protein Heterologous expression of matrix protein increases

transcription but not replication of rabies virus, and
attenuation of matrix protein expression results in rabies
virus with a high transcription rate72. In contrast to these
findings, replacement of the rabies virus matrix protein
of a vaccine strain with that of a bat-derived rabies virus
increased both replication and transcription in a similar
manner 36. Rabies virus matrix protein was also identified
as an important factor in the translation of mRNAs. One
study showed that matrix protein inhibits the transla-
tion of mRNAs with classical 5 end untranslated repeats
but not of mRNAs that contain an internal ribosome entry
site73 through a specific interaction with the eukaryo-
tic translation initiation factor 3 subunit H (eIF3H).
Because rabies virus-derived mRNAs are undistinguish-
able from host cell mRNAs, high concentration of rabies
virus matrix protein should affect not only viral tran-
Nucleoprotein Polymerase Phosphoprotein scription but also translation of viral proteins. However,
Figure 2 | The rabies virus virion. The negative-stranded RNA genome is tightly this could also interfere with the overall strategy of rabies
encapsidated into the nucleoprotein (yellow) and is termed the ribonucleoprotein (RNP). virus to conserve the function of the infected cell, as host
Two other viral proteins are associated with the RNP, the viral polymerase (blue) and the cell protein synthesis would also be affected.
phosphoprotein (orange), which make up the internal core or capsid. The capsid is
engulfed by the host cell-derived membrane, which is associated with two additional
Assembly and budding the final steps. The last steps
viral proteins: the matrix protein (green), which functions as a bridge between the
capsid and the virion membrane, and the single transmembrane glycoprotein, which in the life cycle of rabies virus are assembly of the viral
is organized as a trimer (purple). components and release of virions (known as budding).
For this process, the inner core of the virions (the cap-
sid or RNP) must be engulfed by the host cell mem-
genome65, which are unlikely to contain rabies virus- brane. Budding of rabies virus takes place at the plasma
specific encapsidation sequences. However, when nucle- membrane, but it is unknown how the capsid is trans-
oprotein is expressed alone, it binds equally to any RNA64, ported to the site of budding. In contrast to some other
and non-specific RNA encapsidation is greatly reduced enveloped viruses such as HIV-1, in which the capsid
only when the nucleoprotein is expressed together with protein provides all the functions required for virion
the phosphoprotein. This indicates that the phospho- release (for a review, see ref. 74), both the rabies virus
protein functions as a chaperone for the nucleoprotein matrix protein and glycoprotein play an important part
to reduce non-specific RNA binding 66. Furthermore, in budding. Although there is no absolute requirement
the phosphoprotein has been shown to prevent nucleo- for the glycoprotein, rabies virus is released 30-fold less
protein phosporylation64 by casein kinase II67. Taken efficiently in its absence24. Deletion of the cytoplasmic
together, these findings resulted in the following model. domain of the rabies virus glycoprotein led to a six-
The nucleoproteinphosphoprotein complex interacts fold decrease in the release of virions and revealed a
specifically with viral RNA. During this interaction specific interaction between rabies virus glycoprotein
the nucleoprotein undergoes a conformational change, and matrix protein21. unlike the glycoprotein of VSV,
which enables its phosphorylation64,68. Therefore, the the cytoplasmic domain of rabies virus glycoprotein
amount of the nucleoproteinphosphoprotein complex is required for the incorporation of foreign glycopro-
is one factor responsible for increasing viral replication teins into the virion38,7577. In the absence of rabies virus
and decreasing transcription63. However, the removal matrix protein, viral titres were reduced by as much as
of the phosphorylation site on rabies virus nucleopro- 500,000-fold, although viral protein expression was not
tein impairs both viral replication and transcription69, greatly affected21.
but this is probably an indication that two processes Recently, the structures of the matrix proteins of
are linked. VSV and Lagos bat virus (a rabies virus-related lyssa-
The rabies virus matrix protein has an important role virus) have been solved78. These proteins share a similar
in regulating transcription and replication. A high con- overall structure and the ability to form non-covalent
centration of VSV matrix protein inhibits RNA synthesis linear polymers even though they do not have sequence
in vitro70, and it has been proposed that the association similarity. Self-association is mediated by binding of
of RNP templates and VSV matrix protein results in the a proline-rich motif in the mostly disordered amino-
condensation of the RNP, which then would not be func- terminal region to a pocket in the main globular domain
tional for transcription and replication71. Rabies virus of an adjacent matrix protein. Interestingly, the structure of
matrix protein might also be involved in the condensa- these proteins reveals that the proline-rich self-interaction
Internal ribosome entry site tion of the RNP, but this has not been directly addressed. motifs differ significantly on a molecular level and
rNA sequence that allows
ribosomes to bind to an mrNA
evidence does suggest that, similarly to other rhabdo- notably overlap with known interaction motifs of host
at a position other than the viral matrix proteins, the rabies virus matrix protein proteins in both rabies virus and VSV matrix proteins78.
5 end. is involved in regulating transcription and replication. This important finding indicates that virushost cell

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a Intergenic interaction could be different for these related rhabdo-

region viruses. It also suggests that self-association and interaction
U/ACUUUUUU G(N)124 UUGURRNGA Start
Stop with host cell proteins are regulated.
most enveloped viruses contain late domains, so-
3 N P M G L 5 called because mutations in these domains arrest virus
leRNA morphogenesis at a late stage, when virions are mostly
C N AAAAAAAA formed but fail to bud from the host cell membrane79.
C P AAAAAAAA Late domains mediate efficient budding of virions by
C M AAAAAAAA
C G AAAAAAAA using host cell proteins that normally are involved in
C L AAAAAAAA vacuolar protein sorting (VPS) pathways (for a review,
see ref. 69). The rabies virus matrix protein contains at
Transcription gradient least three motifs that are similar to known late domains:
ASAP (consensus sequence in other viruses is PTAP),
b PPey (consensus sequence in other viruses is PPxy, in
which x denotes any amino acid) and the overlapping
yVPL (consensus sequence in other viruses is yxxL,
in which x denotes any amino acid), forming the motif
PPeyVPL80. All three motifs are located within the
N-terminal region of the matrix protein; this region is
mostly disordered apart from the PPxy, which overlaps
a self-association motif, as described above. Site-directed
1 mutagenesis confirmed the importance of the first pro-
line in the PPey motif and the leucine in the yVPL
2 motif for virus production, whereas the potential role of
the ASAP motif in virus budding has not been addressed
yet. There is also evidence that the ubiquitinproteasome
system is important for virus budding, and in vitro bind-
RNA N P M L G
ing studies indicate that the rhabdovirus PPxy motif can
3
bind ubiquitin ligases81, as has been reported for similar
Transcription motifs in other enveloped viruses.
It remains to be shown to what extent rhabdoviruses
ER use the VPS machinery, as VPS4A, an essential compo-
nent of this pathway, does not seem to be involved in
Translocation
rhabdovirus budding 82. Although no functional interac-
Replication +
tions with components of the VPS pathway have been
shown in vivo, the PPxy motif is probably a hotspot for
interactions with host cell proteins based on information
obtained from the matrix protein crystal structure78 and a
4
promising target for further studies aimed at identifying
Golgi such proteins. Further studies will also need to clarify
whether rhabdovirus budding involves a new pathway
that is distinct from the classical VPS pathway, as has been
suggested for other negative-stranded RNA viruses.

Host cell response to rabies virus infection

Evading the innate immune response. The innate
Figure 3 | Aspects of the rabies virus life cycle. a | Transcription
Nature of the rabies
Reviews virus
| Microbiology
genome. The encapsidated negative-stranded RNA (orange) serves as a template for immune response represents the first defence of the
transcription by the polymerase complex. Transcription starts with a short uncapped host cell against viral infection and involves the secretion
leader RNA (leRNA) from the 3 end of the genomic RNA. This is followed by the of type I interferons (IFNs; including IFN and IFN)
transcription of 5 end-capped (C) and polyadenylated (A) mRNAs, which encode the following the activation of pattern recognition recep-
viral proteins (green). The polymerase complex stops at signal sequence (U/ACUUUUUU), tors, such as TLRs and RNA helicases83. many viruses
ignores the intergenic region of 224 nucleotides and restarts transcription at the have therefore developed mechanisms to escape these
transcription start signal sequence (UUGURRNGA). Successful reinitiation of transcription responses83 (fIG. 5).
at each gene junction does not always occur, therefore transcription is attenuated from It is well established that rabies virus induces innate
the 3 to 5 end59 (this is illustrated by the transcription gradient). b | A simplified rabies
immune responses both in vitro and in vivo8488. Retinoic
virus life cycle in an infected cell can be divided into three different phases. The first
acid-inducible gene I (RIG-I; also known as DDX58) has
phase includes binding and entry into the host cell by endocytosis (step 1), followed by
fusion of the viral membrane and endosome membrane to release the viral genome been shown to be a potent activator of type I IFNs during
(uncoating; step 2). In the second phase, virion components are produced (transcription, rabies virus infection89. RIG-I-mediated type I IFN pro-
replication and protein synthesis; step 3). The last phase of the life cycle is the assembly duction requires the presence of a 5 triphosphate-ended
of the viral components and budding and release of the rabies virus virions (step 4), which RNA89, but not viral replication. However, in contrast to
can start a new round of infection. ER, endoplasmic reticulum. VSV, the role of TLRs for the induction of type I IFNs

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 REVIEWS

Furthermore, the rabies virus phosphoprotein

can inhibit type I and type II IFN signal transduction
pathways62, which indicates that the inhibition of IFN
synthesis by the phosphoprotein is probably not 100%
2
efficient. Additional research indicates that rabies virus
phosphoprotein does not induce the phosphorylation
1
or the degradation of signal transducer and activator
of transcription 1 (STAT1; a transcription factor that
2 is activated by IFN signalling), but instead it interacts
? ? Neuron with and retains STAT1 in the cytoplasm92. moreover,
1 another study showed that only tyrosine-phosphorylated
Capsid STAT1 and STAT2 can be bound by rabies virus phos-
release
phoprotein. This indicates that phosphoprotein binds
STAT1 and STAT2 only following the activation of
2
NCAM
the IFN response, which leads to the phosphorylation
Fusion of STAT1 and STAT2 by janus kinases (fIG. 5). Binding of
Entry into rabies virus phosphoprotein to STAT1 and STAT2
Unknown
neuron? receptor requires 10 carboxy-terminal amino acids in rabies virus
phosphoprotein93.
Synaptic
cleft Of note, rabies virus phosphoprotein is expressed not
only as full-length protein but also in four N-terminal
nAchR truncated forms (phosphoprotein 2phosphoprotein 5)
that are synthesized from internal start codons94. All
Amplification ? phosphoproteins contain a nuclear import sequence94,
Muscle
but phosphoprotein 3, phosphoprotein 4 and phospho-
Figure 4 | rabies virus entry into neurons and protein 5 lack the nuclear export signal found in the
intra-neuronal transport. TheNature Reviews
nicotinic | Microbiology
acetylcholine N-terminus and are retained in the nucleus. As a result,
receptor (nAchR) is located at the postsynaptic muscle the rabies virus phosphoprotein can retain STAT1 in the
membrane. It has been suggested that nAchR enriches cytoplasm and thereby prevent IFN-stimulated growth
rabies virus at the neuromuscular junction or synaptic cleft, factor 3 (ISGF3) from activating the IFN-stimulated
enabling more efficient infection of the connected motor response element (ISRe) in the nucleus95. Interaction
neurons. Other research suggests that initial rabies virus of rabies virus phosphoprotein in the nucleus with the
replication is in muscle cells, indicating that nAchRs might homodimer of STAT1 also interferes with the activation
be used to infect muscle cells. In both cases rabies virus of -activated sequence (GAS)95 (fIG. 5). Recently, it has
enters the neurons using neural cell adhesion molecule
been shown that rabies virus phosphoprotein 3 can also
(NCAM) or another, unknown receptor. Two different
mechanisms have been proposed for the transport of facilitate the interaction of STAT1 and microtubules,
rabies virus through the axon to the cell body: the transport which prevents STAT1 nuclear import. This is another
of either the rabies virus capsid or the whole rabies virus illustration of the ways in which viral proteins can affect
virion within the vesicle. The evidence favours the IFN signalling on multiple levels96.
transport of intact virions. Last, there is evidence that the phosphoprotein retains
the IFN-induced promyelocytic leukaemia (PmL) pro-
tein in the cytoplasm97. This protein is found in PmL
following rabies virus infection is less clear. One study nuclear bodies, which have many possible functions in
showed that TLR3 is upregulated in rabies encephalitis90, nuclear trafficking, viral defence mechanisms and apop-
and the same laboratory recently found that TLR3 has tosis98. How PmL nuclear bodies function in viral infec-
an important function in the formation of Negri bod- tion is still not understood, but the binding and retention
ies54. However, a role for TLR3 in the induction of pro- of PmL by rabies virus phosphoprotein indicates that
inflammatory responses, as is the case for TLR4, TLR7 PmL may have an antiviral function97,99.
and TLR9, still needs to be shown.
expression of IFN greatly reduces rabies virus Rabies virus and apoptosis. many neurotropic viruses
pathogenesis and viral replication but not its immu- use apoptosis as a mechanism of neuropathogenic-
nogenicity 91. Therefore, it might be expected that ity 100. The induction of apoptosis by rabies virus
rabies virus suppresses such host defence mecha- has been a controversial topic, but increasing evi-
nisms. Indeed, the rabies virus phosphoprotein inter- dence indicates that pathogenic rabies virus strains
feres with the type I IFN responses by preventing the do not induce apoptosis 101. It was initially thought
phosphorylation of IFN regulatory factor 3 (IRF3) by that apoptosis had an important role in rabies virus
TANK-binding kinase 1 (ref. 61) (fIG. 5). The expression infection 102,103; for example, induction of apoptosis
level of rabies virus phosphoprotein is crucial for the has been detected in cultured rat prostatic adeno-
prevention of type I IFN responses, and recombinant carcinoma cells after infection with a CVS strain of
rabies virus expressing low levels of phosphoprotein rabies virus102. However, rabies virus strains that are
fails to suppress the production of IFN61. propagated by passage in animals or tissue culture

NATuRe ReVIeWS | Microbiology VOLume 8 | jANuARy 2010 | 57

2010 Macmillan Publishers Limited. All rights reserved

REVIEWS

P NES NLS phenotype compared with pathogenic rabies virus

H2N COOH strains. This is supported by proteomic profiling
showing that an attenuated rabies virus strain mostly
P1 P2 P3 P4 P5
Type I IFN Type II IFN upregulated the production of pro-apoptotic proteins,
IFNAR1 IFNAR2 IFNGR1 whereas a pathogenic strain affected the expression of
IFNGR2
proteins involved in ion homeostasis, docking and
P P P ssRNA TYK2 JAK2
fusion of synaptic vesicles108. One exception to this
JAK2
P P P rule is the finding that only infection by the patho-
P
RIG-I
P P
JAK1 genic rabies virus strain CVS results in the apoptosis
of T cells owing to early upregulation of CD95 ligand
STAT1 P STAT2 (also known as FASL) by infected neurons85. When
TANK P STAT1 P STAT1 detected, induction of apoptosis during rabies virus
P infection can occur by both caspase-dependent and
TBK1 IKK P caspase-independent pathways109111, and overexpres-
IRF3 P sion of the anti-apoptotic protein B cell lymphoma 2
IRF3 P P P
IRF3 P RV prevented apoptosis in AT3 and jurkat cells after rabies
RV P P
RVP IRF9 P RV virus infection102,110. In addition, expression of a pro-
RV P
apoptotic protein, such as cytochrome c, can reduce
rabies virus pathogenicity while increasing its immu-
Cytoplasm
nogenicity 112. Interestingly, it was shown that high lev-
IRF9
ISGF3 P P
els of rabies virus glycoprotein seem to be a key factor
Nucleus P P in the induction of apoptosis51,113, which could explain
RVP RVP RVP RVP why apoptosis is mostly detected during infection with
IRF3 highly attenuated rabies virus strains, which express
P IFNB
ATF2 NF-B IRF3 P much higher levels of viral proteins than pathogenic
ISRE GAS
strains114. It is interesting to note that there is now
clear evidence that rabies virus can actively promote
Figure 5 | rabies virus inhibition of the innate immune response. Rabies
Nature Reviews virus
| Microbiology
phosphoprotein (RVP) antagonizes interferon (IFN) responses by blocking the host cell neuronal survival that depends on an interaction with
response to pathogen-associated molecular patterns and type I and II IFN signalling. cellular partners recruited by the PDZ-binding site
For example, the 5 triphosphate-ended RNA of rabies virus is detected by retinoic of the glycoprotein cytoplasmic domain, and silenc-
acid-inducible gene I (RIG-I), which signals through TRAF-family-member-associated ing of certain host cell phosphatases abrogates rabies
NF-kB activator (TANK) and the TANK-binding kinase 1 (TBK1) IB kinase (IKK) virus-mediated apoptosis115.
complex to induce the phosphorylation of IFN regulatory factor 3 (IRF3). In summary, most evidence indicates that patho-
Phosphorylated IRF3 dimerizes and is transported to the nucleus to induce the genic rabies virus does not induce apoptosis and that
transcription of IFNB in conjunction with activating transcription factor 2 (ATF2) and the induction of apoptosis actually reduces rabies virus
nuclear factor-B (NF-B). In addition, some studies show IFN induction following
pathogenesis. Induction of apoptosis depends largely
signalling by Toll-like receptor 3 (TLR3; not shown); the function of other TLRs still
on the expression levels and the amino acid sequence
needs to be elucidated. RVP inhibits the induction of IFN by preventing the
phosphorylation of IRF3, thus retaining IRF3 in the cytosol. RVP can also inhibit type I of the rabies virus glycoprotein113. Whether this is the
(IFN and IFN) and type II (IFN) IFN signal transduction pathways. Following the consequence of differences in protein stability or a
triggering of type I and type II IFN receptors (IFNRs), signal transducer and activator lower propensity to misfold and induce endoplasmic
of transcription 1 (STAT1) is phosphorylated by Janus kinase (JAK). RVP binds to reticulum stress or due to other factors has yet to be
phosphorylated STAT1, preventing its translocation to the nucleus and the subsequent determined. It also remains to be shown whether
antiviral transcriptional response. In the nucleus, shorter versions of RVP bind to STAT1 pathogenic strains limit apoptosis solely by reducing
and STAT2 heterodimers complexed with IRF9 (forming the IFN-stimulated growth viral replication and transcription and changing the
factor 3 (ISFG3) complex) and STAT1 homodimers. This prevents transcriptional amino acid sequence of their glycoprotein or whether
activation of the IFN-stimulated response element (ISRE) and -activated sequence
they use an additional direct mechanism to prevent
(GAS) and type I and type II IFN-dependent immune response. Different versions of
RVP are produced depending on the transcriptional start site used (see inset).
apoptosis.
Phosphoprotein 3 (P3), P4 and P5 lack the nuclear export signal (NES) and accumulate
in the nucleus. TYK2, tyrosine kinase 2; NLS, nuclear localization signal. Inducible nitric oxide synthase. Studies have examined
the mRNA levels of inducible nitric oxide synthase
(iNOS) in rat brains after infection with different neuro-
do not always maintain a pathogenic phenotype104. tropic viruses, including rabies virus. In contrast to
The highly attenuated rabies virus strain eRA, but herpes simplex virus 1 and Borna disease virus, iNOS
not the pathogenic CVS strain, induces apoptosis in mRNA was only detected in some animals infected
mouse and human lymphocytes105. Similarly, the more with rabies virus116. moreover, the detection of only
attenuated isolate CVS-B2C, but not the pathogenic minimal inflammation in all rabies virus-infected
isolate CVS-N2c, induces apoptosis in primary mouse animals provides evidence that iNOS induction is
neurons106. Furthermore, there is evidence that apop- not likely to be responsible for the observed neu-
tosis does not have a role in human rabies10,107. These ropathology of rabies virus. Therefore, it is of inter-
findings highlight that attenuated rabies viruses (for est to determine whether the induction of iNOS can
example, vaccine strains) display a more pro-apoptotic decrease rabies virus pathogenicity. In support of this

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2010 Macmillan Publishers Limited. All rights reserved

 REVIEWS

hypothesis, Phares et al.117,118 showed that the changes study of the interaction of rabies virus phosphoprotein
in the permeability of the brainblood barrier, a with host cell proteins that are involved in the innate
well-known effect of iNOS, are indeed beneficial for immune response is a good example for such progress.
resisting rabies virus infection and can promote viral Only when these basic processes of virushost cell
clearance. This is in contrast to the findings of ubol interaction are further understood can new therapeutic
et al.119, who showed that inhibition of iNOS produc- targets be identified.
tion delays death of mice after rabies virus infection. Another advancement in rabies virus research could
However, the two studies used different rabies virus be a more restricted use of viral strains. Although
strains (namely a highly pathogenic and attenuated it is justified and necessary to use different primary
strain by Phares et al. and fixed strain by ubol et al.) rabies virus isolates to test, for example, the efficiency
and inoculation routes119,120. most results indicate that of rabies virus vaccines, the use of such strains to
iNOS induction is essential for permeabilizing the study the biology of rabies virus needs to be revisited.
brainblood barrier and allowing entry of the neces- In the case of rabies virus, the choice of a particular
sary effector cells to clear the virus. It is worth not- viral strain is important and should be dictated by
ing that iNOS induction is rarely detected following the studys hypothesis. For example, an attenuated
infection with pathogenic rabies viruses but is detected strain of rabies virus can be an excellent model sys-
following infection with attenuated ones87,117,118,120122. tem to study apoptosis in general but it might not be
a good model system to study rabies virus transport
Summary and outlook or pathogenesis. Three different infectious clones of
Rabies virus has a complex life cycle, and we have only rabies virus vaccine strains are currently available, and
just begun to appreciate its interactions with host cells two of them are almost identical15,123,124. In addition,
on a molecular level. Great progress has been made in infectious rabies virus cDNA was generated for two
understanding the functions of the viral proteins in highly pathogenic rabies virus strains, silver-haired bat
viral replication, transcription, assembly and budding variant of rabies virus125 and a fixed pathogenic rabies
owing to the ability to directly manipulate the viral virus (CVS-N2c; C.W., unpublished observations).
genome. Studies investigating the interaction with The use of such viruses and targeted mutations in their
and use of host cell proteins by rabies virus are at an genomes might help to better compare the findings of
early stage but are progressing rapidly. The detailed different studies.

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FURTHER INFORMATION
WHO Fact Sheet No. 99: http://www.paho.org/common/
nitric oxide synthase inhibition delays death of rabies monophosphoryl lipid A (MPL). Vaccine 18,
10591066 (2000). Display.asp?Lang=E&RecID=10209
virus-infected mice. J. Med. Microbiol. 50, 238242
135. Cenna, J. et al. Immune modulating effect by a Matthias J. Schnells homepage: http://jefferson.edu/jmc/
(2001).
phosphoprotein-deleted rabies virus vaccine vector microbiology/vaccine_center
120. Roy, A. & Hooper, D. C. Lethal silver-haired bat rabies
virus infection can be prevented by opening the blood- expressing two copies of the rabies virus All links Are AcTive in The online pdf
brain barrier. J. Virol. 81, 79937998 (2007). glycoproteins. Vaccine (2008).

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