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AMB 222: GENERAL MICROBIOLOGY II

VIRAL STRUCTURE AND REPLICATION BY DR. V. N. ANAKWENZE

Viruses are noncellular genetic elements that use a living cell for their replication and have
an extracellular state. Viruses are ultramicroscopic particles containing nucleic acid
surrounded by protein, and in some cases, other macromolecular components such as a
membranelike envelope.

Outside the host cell, the virus particle is also known as a virion. The virion is metabolically
inert and does not grow or carry on respiratory or biosynthetic functions.

Viruses vary considerably in size and shape. The smallest viruses are about 0.02 μm (20
nanometers), while the large viruses measure about 0.3 μm (300 nanometers). Smallpox
viruses are among the largest viruses; polio viruses are among the smallest.

Viral structure. Certain viruses contain ribonucleic acid (RNA), while other viruses have
deoxyribonucleic acid (DNA). The nucleic acid portion of the viruses is known as the
genome. The nucleic acid may be single-stranded or double-stranded; it may be linear or a
closed loop; it may be continuous or occur in segments.

The genome of the virus is surrounded by a protein coat known as a capsid, which is formed
from a number of individual protein molecules called capsomeres. Capsomeres are arranged
in a precise and highly repetitive pattern around the nucleic acid. A single type of capsomere
or several chemically distinct types may make up the capsid. The combination of genome and
capsid is called the viral nucleocapsid.

A number of kinds of viruses contain envelopes. An envelope is a membranelike structure

that encloses the nucleocapsid and is obtained from a host cell during the replication process.
The envelope contains viral-specified proteins that make it unique. Among the envelope
viruses are those of herpes simplex, chickenpox, and infectious mononucleosis.

The nucleocapsids of viruses are constructed according to certain symmetrical patterns. The
virus that causes tobacco mosaic disease, for example, has helical symmetry. In this case, the
nucleocapsid is wound like a tightly coiled spiral. The rabies virus also has helical symmetry.
Other viruses take the shape of an icosahedron, and they are said to have icosahedral
symmetry. In an icosahedron, the capsid is composed of 20 faces, each shaped as an
equilateral triangle (Figure 1 ). Among the icosahedral viruses are those that cause yellow
fever, polio, and head colds. Some viruses do not neatly fit into either of the two previous
categories because they are so unusual in design or components, so there is a third category
known as complex viruses. Examples include the poxvirus with a brick-shaped exterior and a
complicated internal structure, as well as bacteriophage with tail fibers attached to an
icosahedral head.
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Figure 1

An array of viruses. (a) The helical virus of rabies. (b) The segmented helical virus of
influenza. (c) A bacteriophage with an icosahedral head and helical tail. (d) An enveloped
icosahedral herpes simplex virus. (e) The unenveloped polio virus. (f) The icosahedral
human immunodeficiency virus with spikes on its envelope.

The envelope of certain viruses is a lipid bilayer containing glycoproteins embedded in the
lipid. The envelope gives a somewhat circular appearance to the virus and does not contribute
to the symmetry of the nucleocapsid. Projections from the envelope are known as spikes. The
spikes sometimes contain essential elements for attachment of the virus to the host cell. The
virus of AIDS, the human immunodeficiency virus, uses its spikes for this purpose.
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Bacteriophages are viruses that multiply within bacteria. These viruses are among the more
complex viruses. They often have icosahedral heads and helical tails. The virus that attacks
and replicates in Escherichia coli has 20 different proteins in its helical tail and a set of
numerous fibers and “pins.” Bacteriophages contain DNA and are important tools for viral
research.

Viral Replication

During the process of viral replication, a virus induces a living host cell to synthesize the
essential components for the synthesis of new viral particles. The particles are then assembled
into the correct structure, and the newly formed virions escape from the cell to infect other
cells.

The first step in the replication process is attachment. In this step, the virus adsorbs to a
susceptible host cell. High specificity exists between virus and cell, and the envelope spikes
may unite with cell surface receptors. Receptors may exist on bacterial pili or flagella or on
the host cell membrane.

The next step is penetration of the virus or the viral genome into the cell. This step may
occur by phagocytosis; or the envelope of the virus may blend with the cell membrane; or the
virus may “inject” its genome into the host cell. The latter situation occurs with the
bacteriophage when the tail of the phage unites with the bacterial cell wall and enzymes open
a hole in the wall. The DNA of the phage penetrates through this hole.

The replication steps of the process occur next. The protein capsid is stripped away from the
genome, and the genome is freed in the cell cytoplasm. If the genome consists of RNA, the
genome acts as a messenger RNA molecule and provides the genetic codes for the synthesis
of enzymes. The enzymes are used for the synthesis of viral genomes and capsomeres and the
assembly of these components into new viruses. If the viral genome consists of DNA, it
provides the genetic code for the synthesis of messenger RNA molecules, and the process
proceeds.

In some cases, such as in HIV infection (as discussed below), the RNA of the virus serves as
a template for the synthesis of a DNA molecule. The enzyme reverse transcriptase catalyzes
the DNA's production. The DNA molecule then remains as part of the host cell's chromosome
for an unspecified period. From this location, it encodes messenger RNA molecules for the
synthesis of enzymes and viral components.

Once the viral genomes and capsomeres have been synthesized, they are assembled to form
new virions. This assembly may take place in the cytoplasm or in the nucleus of the host cell.
After the assembly is complete, the virions are ready to be released into the environment
(Figure 2 ).
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Figure 2

A generalized representation of the replication of two viruses. Replication of a DNA virus is

shown in (1); replication of an RNA virus is displayed in (2).

For the release of new viral particles, any of a number of processes may occur. For example,
the host cell may be “biochemically exhausted,” and it may disintegrate, thereby releasing the
virions. For enveloped viruses, the nucleocapsids move toward the membrane of the host cell,
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where they force themselves through that membrane in a process called budding. During
budding, a portion of cell membrane pinches off and surrounds the nucleocapsid as an
envelope. The replication process in which the host cell experiences death is called the lytic
cycle of reproduction. The viruses so produced are free to infect and replicate in other host
cells in the area.

Lysogeny. Not all viruses multiply by the lytic cycle of reproduction. Certain viruses remain
active within their host cells for a long period without replicating. This cycle is called
the lysogenic cycle. The viruses are called temperate viruses, or proviruses, because they
do not bring death to the host cell immediately.

In lysogeny, the temperate virus exists in a latent form within the host cell and is usually
integrated into the chromosome. Bacteriophages that remain latent within their bacterial host
cell are called prophages. This process is a key element in the recombination process known
as transduction.

An example of lysogeny occurs in HIV infection. In this case, the human immunodeficiency
virus remains latent within the host T-lymphocyte. An individual whose infection is at this
stage will not experience the symptoms of AIDS until a later date.

Viral Cultivation and Physiology

Viruses can be cultivated within suitable hosts, such as a living cell. To study bacteriophages,
for example, bacteria are grown in a suitable growth medium; then bacteriophages are added.
The bacteriophages multiply within the bacteria and increase their numbers substantially.
Animal and plant viruses are cultivated in cell cultures. A cell culture is prepared by
encouraging cell growth outside the animal or plant source. The cells are kept alive in a
suspension of growth factors within a Petri dish. A thin layer of cells, or monolayer, is then
inoculated with viruses, and replication takes place. Fertilized eggs and living animals can
also be used to cultivate viruses.

For research study, viruses can be cultivated in large volumes by inoculations to tissue
culture systems. After a time, the cells are degenerated, and viruses are harvested. The viral
particles are concentrated by precipitation methods and purified by repeated centrifugations.
Highly purified viruses can be obtained by crystallization and concentration under established
conditions.

Viral measurements. Viruses are generally too small to be seen under the light microscope,
and an electron microscope is usually necessary to make them visible. Although viruses can
be quantified by observation, it is also possible to determine their number in terms of virus
infectious units, each of which is the smallest unit that causes a detectable effect when
viruses infect a susceptible host. Virus infectious units are expressed per volume of fluid.

One method for determining virus infectious units is by the plaque assay. The plaque assay
is performed by cultivating viruses on a “lawn” of host cells and noting the presence of clear
areas where viruses have replicated and destroyed the cells.

Another way of determining virus infectious units is by cultivating viruses in living animals
and determining which dilution of virus is lethal to the animals. The end-point dilution can
be calculated by this method.
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Antiviral agents. The antibiotics normally used to treat bacterial disease cannot be used to
inactivate viruses because viruses do not perform the biochemical functions that antibiotics
interfere with. For example, penicillin is used to interrupt the synthesis of the bacterial cell
wall, but viruses have no cell wall.
However, there are several nucleotide analog drugs that interfere with viral
replication.Acyclovir, for example, is used against herpes viruses because this drug prevents
the synthesis of DNA during viral replication. A drug called azidothymidine (AZT) is used
for patients with HIV infection because this drug also prevents the synthesis of DNA. A drug
called ganciclovir is used against cytomegaloviruses, and amantadine is useful against
influenza viruses.
Interferon, a naturally produced antiviral agent approved for certain uses, is a group of
proteins produced by host cells after they have been infected by viruses. The interferons do
not protect the host cell, but they do provide protection to neighboring cells against viral
replication. Interferons can be produced by genetic engineering methods.

Viral vaccines.
Protection against viral disease can be rendered by using a viral vaccine. Viral vaccines can
be composed of inactivated or attenuated viruses. Inactivated viruses (“dead viruses”) are
unable to replicate in host cells because of some chemical or physical treatment. The Salk
vaccine against polio and the yellow fever vaccine are examples.

Attenuated viruses (“live viruses”) are weakened viruses that replicate at a very slow rate in
host cells and generally do not produce any symptoms of disease when inoculated to humans.
Attenuated viruses are used in the Sabin polio vaccine and in the vaccines against measles
and rubella. The most contemporary vaccines are composed of viral proteins produced
by genetic engineering methods. The vaccine for hepatitis B is an example of this type of
vaccine.
Viral inactivation. Virus particles are composed of nucleic acid, protein, and in some cases,
a lipid envelope. As such, the viruses are susceptible to normal inactivation by chemical
substances that react with any of these organic compounds. Such things as chlorine, iodine,
phenol, detergents, and heavy metals rapidly inactivate viruses. In addition, viruses are
destroyed by heating methods used for other microorganisms, and they are very susceptible to
the effects of ultraviolet light. Filters can be used to remove viruses from fluids so long as the
filter pores are small enough to trap viral particles.

OTHER ACELLULAR INFECTIOUS AGENT BY MRS. E. UDENWEZE

Prions
Prions are proteinaceous infectious particles, which can transmit a disease. In 1982, Stanley
Prusiner discovered that scrapie disease of sheep was caused by proteinaceous infectious
particles. He called the proteinaceous particle a prion. A prion is a misfolded rogue form of a
normal protein (PrPc) found in the cell. This rogue prion protein (PrP sc), which may be caused
by a genetic mutation or occur spontaneously, can be infectious, stimulating other
endogenous normal proteins to become misfolded. The prion does not contain any nucleic
acid. It contains an aggregate of hydrophobic glycoprotein, resistant to protease.
Unlike conventional viruses, the prions have no virion structure or genomes and evoke no
immune response in the infected host. They are extremely resistant to inactivation by heat,
disinfectants, and radiation.
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Properties of the Prions

 Prions like viruses are filterable. They are smaller than smallest viruses
 They are obligate intracellular parasite
 They apparently lack any virion structure or genome.
 Prions, unlike viruses, are unusually resistant to heat at 80°C,ultraviolet radiation, and
disinfectants, such as formaldehyde however ,are inactivated by phenol, ether,
sodium hydroxide, and hypochlorite..
 They do not elicit any specific immune response in the infected host
 Prions cannot be cultivated in any tissue culture or isolated in any experimental
animal models
Replication of prion
Proteins similar to prion proteins in its amino acid composition is synthesized in normal
uninfected cells. Prion therefore multiply by converting the normal host proteins into prion
proteins by changing the folding properties of the protein (PrP) . PrP exists in two
forms, PrPc, the normal form of the protein, and PrPsc, the infectious form. Once introduced
into the body, the PrPsc contained within the prion binds to PrPc and converts it to PrPsc

Diseases caused by prions

The prions are causative agents of slow viral infections. The diseases caused by prions are a
large group of related neurodegenerative conditions, which affect both humans and animals.
These diseases belong to a family of diseases known as the transmissible spongiform
encephalopathies (TSEs).
There are six human TSEs caused by prions:
1. Kuru
2. Creutzfeldt–Jakob disease (CJD),
3. variant CJD (vCJD),
4. Gerstmann–Straussler–Scheinker (GSS) syndrome,
5. fatal familial insomnia (FFI),
6. sporadic fatal insomnia.
Animal TSEs caused by prions include
1. scrapie and visna (diseases of sheep)
2. Bovine spongiform encephalopathy (BSE; mad cow disease),
3. transmissible mink encephalopathy
4. Chronic wasting disease of deer, mule, and elk.

Viroids
Viroids are protein-free fragments of single-stranded, circular RNA molecule that are
infectious pathogen. Viroids were first discovered and named by the plant pathologist
Theodor Otto Diener in 1971. The first viroid identified was Potato Spindle Tuber Viroid
(PsTVd)

Structure of Viroid:
The viroid is made up of a protein-free, low molecular weight (75000-125000 daltons), naked
single-stranded RNA.
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The single-stranded RNA is usually folded to form a circular structure that exist as rod-like
shape to complementary base pairing at intervals. The number of nucleotides is about 250-
400 in each RNA molecule.
The folded RNA possesses the Five regions – Left terminal domain (T L), Pathogenicity
domain (P domain), Central Conserved region (CCR), Variable domain (V domain), and
Right terminal domain (TR).
RNA does not code any protein due to the absence of initiation of the codon.

Structure of Viroid

Some of the characteristic features of viroid

1. Viroids contain only RNA capable of infecting a cell
2. They are smaller than viruses
3. They are replicated by host cell DNA –dependent RNA polymerases
4. They are obligate intracellular parasites that infect only the plants.
5. Viroids RNA does not code for any protein
6. They do not have capsid (protein coat) The low-molecular-weight RNA is resistant to
heat and organic solvents but sensitive to nucleases
7. Viroids are mainly classified into two families namely Pospiviroidae- nuclear viroids
and Avsunviroidae- chloroplastic viroids.
Transmission of viroids
Viroids only infect plants, and infectious viroid can be transmitted to new plant hosts by
aphids, by cross contamination following mechanical damage to plants as a result of
horticultural or agricultural practices, or from plant to plant by leaf contact.
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Replication of viroids
Viroid hijack the cellular machinery present in plant cells to replicate new copies of
themselves. Replication of viroid takes place in the nucleus or chloroplast of the host cell.
Viroid is replicated by a host cell enzyme called a DNA-dependent RNA polymerase. This
enzyme normally functions in the host to synthesize RNA using DNA as the template during
transcription. The host polymerase synthesizes a complementary RNA molecule, which then
serves as the template for synthesis of new viroid RNAs.
The replication by the rolling circle mechanism which occur in three steps
1. production of longer-than-unit strands catalyzed by either the nuclear RNA
polymerase II or a nuclear-encoded chloroplastic RNA polymerase (enzyme that
catalyses synthesis of mRNA from DNA)
2. cleavage to unit-length
3. circularization catalyzed by RNA ligase
.
Types of Viroids
The 30 known viroids belong to two families- Pospiviroidae and Avsunviroidae.
Pospiviroidae: Members of the pospiviroidae are named after the potato spindle tuber
viroid. They have a rod-like structure with a small single-stranded region. They also have a
central conserved region and replicate in the nucleus.
Avsunviroidae: is named after avocado sunblotch viroid. This viroid also has rod-like
branched regions. However, this viroid lacks a central conserved region and replicates in the
chloroplast. RNA molecules are working ribozymes, and this activity is crucial for
replication.
Viroid diseases
 Viroids are known to infect plants only .Symptoms of viroid infection in plant include
stunted growth, deformation of leaves and fruits, stem necrosis and death. There are
over 20 plant diseases caused by the two families of viroid. They include; Potato
spindle tuber viroid disease, Tomato chlorotic dwarf viroid disease, Citrus exocortis
viroid, Tomato apical stunt viroid, Avocado sunblotch viroid, Peach latent mosaic
viroid, Eggplant latent viroid etc

Satellites
A satellite is a sub viral agent that depends on the coinfection of a host cell with a helper
virus for its replication and other essential functions. Satellite genome may be single –
stranded RNA or DNA or circular RNA and are replicated by enzymes provided by the
helper virus.

Features of Satellites
 They depend on helper viruses, at least for replication;
 They are not part of the helper viral genome and are often not required for the
infection cycle of their helper viruses
 Satellites share little or no nucleotide sequence similarity with their cognate helper
viruses
 They can modulate, attenuate or exacerbate the symptoms caused by their
cognate helper viruses
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Satellites are further divided into three types:

 Satellite viruses
 Satellite RNAs
 Satellite DNAs.
Satellite viruses
Satellite viruses are particles that contain nucleic acid genomes that encode structural
proteins that encapsidate their genetic material but rely on helper virus for replication and
transmission of disease. Their structural proteins are distinct from that of their helper
viruses. Satellite viruses may infect plants, animals, or bacteria.
There are only four known plant satellite viruses, Tobacco necrosis satellite virus,
panicum mosaic satellite virus, maize white line satellite virus and Tobacco mosaic
satellite virus. The satellite viruses are named after their associated helper virus.
Adenovirus-associated (AAV) satellite virus particles are found in humans and
domesticated animals

Satellite nucleic acids

Satellite nucleic acids, in contrast, do not encode their own structural proteins, but instead
are encapsulated by proteins encoded by their helper viruses.

Satellite RNA (satRNAs)

These are short RNA molecules that depend on cognate helper viruses for replication,
encapsidation, movement, and transmission. They share little or no sequence homology
with the helper viruses. Satellite RNA genomes range in length from 220-1500
nucleotides, and have been placed into one of three sub-groups
1; large satellite RNAs : are 800-1500 nucleotide linear molecules with a single open
reading frame encoding at least one non-structural protein. Examples include :
Arabis mosaic virus satellite RNA, Tomato black ringspot satellite RNA, Bamboo
mosaic virus Satellite RNA, Grapevine fan leaf virus satellite RNAetc
2 ; Small linear satellite RNAs are linear, less than 800 nucleotides long and do not
encode protein. Examples include; cucumber mosaic virus satellite RNA, Groundnut
rosette virus satellite RNA, Tomato bushy stunt virus satellite RNA, Peanut stunt virus
satellite RNA etc
3 Circular satellite RNAs ( Virusoids ) are 350-400 nucleotide long circles without an
open reading frame. Examples include: Cereal yellow dwarf virus satellite RNA, Tobacco
ringspot virus satellite RNA etc.

Satellite DNAs
Satellite DNAs are usually circular DNAs of about 1.3 kb that encode a nonstructural
protein known as βC1. The satellite DNAs, also known as DNAβ components, are
associated with begomoviruses .Another shorter, noncoding form of satellite DNAs has
been found associated with Tomato leaf curl virus

Virusoids
Virusoids are circular single-stranded RNA(s) dependent on viruses for replication and
encapsidation. The genome of virusoids consist of several hundred (200–400) nucleotides
and does not code for any proteins. They are packaged as passengers into the virus
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capsids. They do not interfere with the replication of their helper virus. Virusoids belong
to a larger group of infectious agents called circular satellite RNAs, which can be found
in bacteria, plants, fungi, invertebrates, and vertebrates.

Replication
RNA replication of virusoids is similar to that of viroids (rolling mechanism) but, unlike
viroids, virusoids require that the cell also be infected with a specific “helper” virus. Once
the helper virus enters the host cell, the virusoids are released and can be found free in
plant cell cytoplasm, where replicate by rolling mechanism. The helper virus undergoes
typical viral replication independent of the activity of the virusoid

Diseases caused by Virusoid

There are currently only five described types of virusoids and their associated helper
viruses.
Barley yellow dwarf disease caused by barley yellow dwarf virus satellite RNA and a
helper virus
Tobacco ring spot disease caused by tobacco ring spot virus satellite RNA and a helper
virus: Nepovirus.
Clover mottle disease caused by Clover mottle virus satellite RNA and a helper
virus :Sobemovirus.
These virusoids can modify the symptoms of infection by their helper virus and are
spread by vegetative propagation, within seeds or by direct inoculation either by insects
or man.
Hepatitis delta virus (HDV) that infect human have recognized as called hepatitis delta
virusoid. HDV helper virus is the hepatitis B virus (HBV). Coinfection with HBV and
HDV results in more severe pathological changes in the liver.
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