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Introduction to Viruses:
A virus is a biological agent that reproduces inside the cells of living hosts. When
infected by a virus, a host cell is forced to produce many thousands of identical copies of
the original virus, at an extraordinary rate. Unlike most living things, viruses do not have
cells that divide; new viruses are assembled in the infected host cell. Over 2,000 species
of viruses have been discovered. Notable human diseases caused by viruses include
SARS, influenza and hepatitis C
A virus consists of two or three parts: all viruses have genes made from either DNA or
RNA, long molecules that carry the genetic information; all have a protein coat that
protects these genes; and some have an envelope of fat that surrounds them when they are
not within a cell. Viruses vary in shape from the simple helical and icosahedral to more
complex structures. Viruses are about 100 times smaller than bacteria, and it would take
30,000 to 750,000 of them, side by side, to stretch to 1 centimeter
Life-cycle of Viruses:
When a virus infects a cell, the virus forces it to make thousands more viruses. It does
this by making the cell copy the virus's DNA or RNA, making viral proteins, which all
assemble to form new virus particles.
There are six basic, overlapping stages in the life cycle of viruses in living cells.
Attachment: the binding of the virus to specific molecules on the surface of the
cell. This specificity restricts the virus to a very limited type of cell. For example,
the human immunodeficiency virus (HIV) infects only human T cells, because its
surface protein, gp120, can only react with CD4 and other molecules on the T
cell's surface. Plant viruses can only attach to plant cells and cannot infect
animals. This mechanism has evolved to favor those viruses that only infect cells
in which they are capable of reproducing.
Penetration follows attachment; viruses penetrate the host cell by endocytosis or
by fusion with the cell.
Uncoating: happens inside the cell when the viral capsid is removed and
destroyed by viral enzymes or host enzymes, thereby exposing the viral nucleic
acid.
Replication of virus particles is the stage where a cell uses viral messenger RNA
in its protein synthesis systems to produce viral proteins. The RNA or DNA
synthesis abilities of the cell produce the virus's DNA or RNA.
Assembly takes place in the cell when the newly created viral proteins and
nucleic acid combine to form hundreds of new virus particles.
Release occurs when the new viruses escape or are released from the cell. Most
viruses achieve this by making the cells burst, a process called lysis. Other viruses
such as HIV are released more gently by a process called budding.
Viral Genetics:
Viral genetics, the study of the genetic mechanisms that operate during the life cycle of
viruses, utilizes biophysical, biological, and genetic analyses to study the viral genome
and its variation. The virus genome consists of only one type of nucleic acid, which could
be a single or double stranded DNA or RNA. Single stranded RNA viruses could contain
positive-sense (+RNA), which serves directly as mRNA or negative-sense RNA (-RNA)
that must use an RNA polymerase to synthesize a complementary positive strand to serve
as mRNA. Viruses are obligate parasites that are completely dependent on the host cell
for the replication and transcription of their genomes as well as the translation of the
mRNA transcripts into proteins. Viral proteins usually have a structural function, making
up a shell around the genome, but may contain some enzymes that are necessary for the
virus replication and life cycle in the host cell.
Classification of Viruses:
Viruses are classified in two families depending on whether they have RNA or DNA
genomes and whether these genomes are double or single stranded. Further subdivision
into types takes into account whether the genome consists of a single RNA molecule or
many molecules as in the case of segmented viruses. Four types of bacteriophages are
widely used in biochemical and genetic research. These are the T phages, the temperate
phages typified by bacteriophage lambda, the small DNA phages like M13, and the RNA
phages. Animal viruses are subdivided in many classes and types. Class I viruses contain
a single molecule of double stranded DNA and are exemplified by adenovirus, simian
virus 40 (SV40), herpes viruses, and human papillomaviruses. Class II viruses are also
called parvoviruses and are made of single stranded DNA that is copied in to double
stranded DNA before transcription in the host cell. Class III viruses are double stranded
RNA viruses that have segmented genomes which means that they contain 10-12
separate double stranded RNA molecules. The negative strands serve as template for
mRNA synthesis. Class IV viruses, typified by poliovirus, have single plus strand
genomic RNA that serves as the mRNA. Class V viruses contain a single negative
strand RNA which serves as the template for the production of mRNA by specific virus
enzymes. Class VI viruses are also known as Retroviruses and contain double stranded
RNA genome.
RNA Viruses:
An RNA virus is a virus that has RNA (ribonucleic acid) as its genetic material. This
nucleic acid is usually single-stranded RNA (ssRNA) but may be double-stranded RNA
(dsRNA).
as their genetic material. Positive-sense viral RNA is identical to viral mRNA and thus
can be immediately translated by the host cell. And other RNA viruses are negative in
that they have an "antisense" strand (the paired opposite of the coded information).
Negative-sense viral RNA is complementary to mRNA. Negative-strand or antisensestrand RNA viruses are as opposed to positive-strand or sense-strand RNA viruses. As
such, purified RNA of a positive-sense virus can directly cause infection though it may be
less infectious than the whole virus particle. Purified RNA of a negative-sense virus is not
infectious by itself as it needs to be transcribed into positive-sense RNA.
RHABDOVIRUSES
(RHABDOVIRIDAE)
Example: Rabies virus. The most intensively studied member is vesicular stomatitis virus.
RNA is single stranded, is negative (minus) sense, and codes for 5 proteins. The entire
life cycle occurs in the cytoplasm, RNA polymerase and RNA modification enzymes are
virally-coded and present in the virion itself. There is no early/late division of gene
expression.
copy virion RNA when it is in the nucleocapsid form. This is an advantage in that
genomic RNA is therefore somewhat protected from ribonucleases. There is one
monocistronic mRNA for each of the five virally coded proteins. The mRNAs are capped,
methylated, and polyadenylated. Since this is a cytoplasmic, negative-sense RNA virus,
the enzymes for mRNA synthesis and modification are packaged in the virion.
Translation
Messenger RNAs are translated on host ribosomes and all five viral proteins are made at
the same time. There is no distinction between early and late functions.
RNA replication
RNA replication is the process by which new copies of genome-length RNAs are made.
RNA replication occurs in the cytoplasm and is carried out by the viral RNA polymerase.
The full length plus strand is coated with nucleocapsid protein as it is made (mRNAs are
not coated with this protein, which would interfere with the host protein translation
machinery). The new positive strand is copied into full length minus strand, which is also
coated with nucleocapsid protein as it is made.
New negative strands may now:
be used as templates for the synthesis of more full length plus strands.
be used as templates for the synthesis of more mRNAs.
be packaged into virions. .
Assembly
The newly made RNAs and polymerase complexes are first packed into nucleocapsid
containing G protein and then they are modified in envelop by ribosome on rough
endoplasmic reticulum.
PICORNAVIRUSES (PICORNAVIRIDAE)
These are small (28nm), naked icosahedral viruses (pico=very small). The RNA is singlestranded, plus sense, polyadenylated. It functions as mRNA immediately upon infection.
The entire life cycle occurs in the cytoplasm and there is no division into early and late
gene expression of the genome. Example is poliovirus.
RNA replication
We now have newly made viral proteins to support replication.
1. Viral RNA polymerase copies plus-sense genomic RNA into complementary minussense RNA using VPg (or precursor containing VPg), Viral RNA polymerase (replicase),
and Certain Host proteins. VPg may act as a primer for RNA synthesis, this would
explain why it is at the 5' end of all newly synthesized RNA molecules.
2. New minus sense strands serve as template for new plus sense strands. Again, RNA
polymerase and VPg are needed. VPg is linked to the 5' ends of the new plus sense
strands (again, it probably functions as a primer).
The new plus strand has three alternative fates:
Assembly
When sufficient plus-sense progeny RNA and virion proteins have accumulated,
assembly begins. Particles assemble with VPg-RNA inside and 3 proteins in the capsid
[VP0, 1 and 3]. VP0 is then cleaved to VP2 and VP4 as the virions mature and these
mature virions are infectious. Virions are released following cell lysis. Excess capsids are
formed and inclusion bodies may be seen in the cytoplasm.
ORTHOMYXOVIRUSES (ORTHOMYXOVIRIDAE)
It is also called Influenza virus. There are three groups of influenza virus: A, B and C.
Influenza A virus is most intensively studied and influenza A and B are the most
important in human disease. Influenza viruses are pleomorphic virions (that is, they vary
in shape). They have negative-sense, single-stranded RNA and an RNA genome that is
SEGMENTED. There are eight RNA segments in influenza A. The nucleocapsid is
helical. Virions contain RNA polymerase packaged within the virus particle. These
viruses are enveloped and have two membrane glycoproteins:
Reoviruses have icosahedral symmetry and a multiple layered capsid (inner and outer
capsid). The RNA is double stranded. There are 10-12 segments (depending on the genus
of the Reovirus family). There are some significant differences in the life cycle of
members of the reovirus family and of the rotavirus family. Due to their clinical
importance in humans, focus is on rotaviruses.
ROTAVIRUSES
(rota = wheel (from appearance of virions in the electron-microscope))
Assembly
More proteins are made and eventually the immature capsids bud into the lumen of the
endoplasmic reticulum. In doing so, they acquire a transient envelope which is lost as
they mature. This is a very odd feature of the rotaviruses. Then the release probably
occurs via cell lysis.
Retroviruses:
Retroviruses comprise a large and diverse family of enveloped RNA viruses defined by
common taxonomic denominators that include structure, composition, and replicative
properties. The virions are 80100 nm in diameter, and their outer lipid envelope
incorporates and displays the viral glycoproteins
The shape and location of the internal protein core are characteristic for various genera of
the family. The virion RNA is 712 kb in size, and it is linear, single-stranded, nonsegmented, and of positive polarity. The hallmark of the family is its replicative strategy
which includes as essential steps reverse transcription of the virion RNA into linear
double-stranded DNA and the subsequent integration of this DNA into the genome of the
cell. Retroviruses contain RNA as the hereditary material in place of the more common
DNA. In addition to RNA, retrovirus particles also contain an enzyme called reverse
transcriptase (or RTase) that can both copy minus strand DNA from genomic RNA,
catalyze the synthesis of a complementary plus DNA strand. The resulting double
stranded DNA is integrated in the host chromosome and is transcribed by the host's own
machinery. The resulting transcripts are either used to synthesize proteins or produce new
viral particles. These new viruses are released by budding, usually without killing the
host cell. Both HIV and HTLV viruses belong to this class of viruses.
Retroviruses are broadly divided into two categoriessimple and complex
distinguishable by the organization of their genomes. The retrovirus family is split up into
7 genera: the Alpha retroviruses, the Beta retroviruses, Gamma retroviruses, Delta
retroviruses, Epsilon viruses (all of which used to be classified as one genus, the
oncoviruses), the Lentiviruses (which includes HIV) and the Spumaviruses.
All retroviruses contain three major coding domains with information for virion proteins:
gag, which directs the synthesis of internal virion proteins that form the matrix,
the capsid, and the nucleoprotein structures;
pol, which contains the information for the reverse transcriptase and integrase
enzymes; and
env, from which are derived the surface and transmembrane components of the
viral envelope protein.
An additional, smaller, coding domain present in all retroviruses is pro, which
encodes the virion protease. Simple retroviruses usually carry only this elementary
information, whereas complex retroviruses code for additional regulatory non-virion
proteins derived from multiply spliced messages.
Virus Replication
A detailed summary of the steps of virus replications is as follows:
1)
2)
3)
4)
5)
6)
7)
8)
9)
Human Retroviruses:
Human T-Cell lymphotropic Virus Types I and II:
HTLV- I and II is included in the retroviridae family because of its nucleotide sequence
and genome structure. Morphologically they are named the primate T-cell
leukemia/lymphoma viruses. They are biologically distinct from the human
immunodeficiency viruses in the lentivirus genus. Clinical manifestations of HTLV-I are
linked with the development of adult T-cell leukemia/lymphoma and a progressive
neurological disease called HTLV-I associated myelopathy. HTLV-II is also linked with
leukemia and neurologic disease cases as well.
Human Immunodeficiency Viruses(HIV)
Human immunodeficiency viruses are part of the lentivirus genus. It includes the disease
subtypes HIV-1 and HIV-2, the third and fourth human retroviruses discovered. HIV
enters the host cell through the CD4 molecule and chemokine receptor as a dual receptor
system. The biology of HIV-1 has been highly researched due to the pressing concerns of
an HIV global pandemic and push for vaccine and treatment development.
DNA Viruses:
Dna virus is a virus that has DNA (deoxyribonucleic acid) as its genetic material.
They are usually Large, Icosahedral, enveloped in Lipoproteins, Do not have
polymerase enzymes, and cause Latent infection. Examples include Poxviruses,
Herpesviruses, hepadnaviruses, Hepatitis b.
HERPESVIRUSES :
Larger virions than adenoviruses(180 - 200nm). Larger genome (three to five times) than
adenoviruses. Linear, double-stranded DNA, Enveloped, icosahedral virus (this means
that lipid solvents readily inactivate these viruses)
Early phase
Early transcription (the mRNAs made during this phase are the alpha and beta mRNAs).
Herpes viruses use host RNA polymerase. However, a virion tegument protein (VP16)
enters the nucleus upon infection and is important as part of the transcription factor
complex recognized by the host RNA polymerase. The virus uses host mRNA
modification enzymes.
Initially, alpha-mRNAs are transcribed. These are the immediate early mRNAs and are
exported to the cytoplasm and translated into alpha-proteins. The -proteins translated in
the cytoplasm are transported into nucleus where they enable the beta-promoters to be
used by the host RNA polymerase.
Beta-mRNAs are transcribed by the host RNA polymerase again. (Beta-genes are still
"early" since they are transcribed prior to DNA synthesis. Sometimes alpha-genes are
called "immediate early" and beta-genes are called "early"). Beta proteins are involved in
gene expression regulation. They decrease alpha-gene expression and are needed for
gamma gene expression. They are also involved in various aspects of DNA synthesis; for
example, herpes beta -genes code for a variety of proteins including DNA polymerase,
DNA binding proteins, thymidine kinase, ribonucleotide reductase etc.
Since these beta proteins are virally-coded and not host-coded enzymes, they are
potentially weak links in the virus life cycle and thus promising targets for viral
chemotherapy
Late phase
DNA replication
Herpesviruses code for several proteins, in addition to the DNA polymerase, that are
needed for DNA replication. The precise mechanism of DNA replication is not known.
DNA replication is accompanied by a lot of recombination. The replicated DNA is
present as long concatameric molecules (tandem repeats of the genome linked head-totail). These are cleaved to genome-size lengths when DNA is packaged into the virion.
Late transcription:
By definition, late transcription occurs after DNA replication. Gamma mRNAs are made
and are translated in the cytoplasm. Gamma proteins are predominantly structural. There
is decreased expression of beta genes in the late stage. This is probably due to downregulation of transcription of beta genes, by gamma proteins. In herpes viruses there is no
apparent organization of the genome into blocks for either early or late transcription.
Assembly
Assembly occurs in the nucleus. A capsid is formed and the DNA enters the capsid. The
capsids acquire an envelope by budding through areas of the inner nuclear membrane
which have viral membrane proteins inserted into them. These areas have tegument
proteins associated with the inner face of the inner nuclear membrane. The virus envelope
then fuses with the outer nuclear membrane and the de-enveloped nucleocapisid is
delivered into the cytoplasm, where it acquires a more mature tegument. It then becomes
re-enveloped by budding into Golgi-derived vesicles and is then released.
The late protein required for transcription of immediate early mRNAs in the next round
of infection is packaged in the virion.
Evolution of Viruses:
Viral genomes undergo genetic change by mutation, either spontaneous or induced
mutation, and by recombination. Recombination may be either intramolecular or, among
viruses with divided genomes, by reassortment.
Mutations:
These arise naturally during viral replication: e.g. due to errors by the genome-replicating
polymerase or as a result of the incorporation of tautomeric forms of the bases. Mutation
in RNA viruses may be extremely rapid because RNA is a less thermodynamically stable
molecule than is DNA, many of the mutations which give rise to the diversity seen with
the HIV virus is not a result of the fact that the virus uses an RNA genome, but of the
nature of the polymerase enzyme that the virus uses. There is no proof-reading
mechanism for RNA polymerases, as there is for DNA polymerases. This situation is
compounded in the retroviruses, for there is no proof-reading mechanism for the reverse
transcriptase either. Most of these mutations result in non-viable phenotypes. Whether the
genetic changes lead to emergence of an altered phenotype depends on natural selection,
which may occur within the infected cell, during spread of virus in the body, or the
transmission of the virus from one host to the next. Mutations can manually b introduced
by Agents acting directly on bases, e.g. nitrous acid, Agents acting indirectly, e.g. base
analogs which mis-pair more frequently than normal bases or even by Agents such as UV
light or X-rays
References:
www.Stanford.edu
www.ncbi.nlm.nih.gov
http://pathmicro.med.sc.edu/mhunt/dna1.htm
http://science.jrank.org/pages/7190/Viral-Genetics.html
NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.
Coffin JM, Hughes SH, Varmus HE, editors. Retroviruses. Cold Spring Harbor (NY): Cold Spring
Harbor Laboratory Press; 1997.
Reverse transcription of retroviral genomic RNA yields doublestranded DNA that is integrated into the host genome to form a
provirus. Transcription of proviral DNA recreates the full-length viral
RNA genome, and subgenomic-sized RNA molecules are generated by
RNA processing. All RNA products serve as templates for the
production of viral proteins.
The formation of the provirus is a unique strategy among animal
viruses and places retroviruses among the classes of mobile elements
known as retrotransposons. In the DNA intermediate stage, the virus
mimics a cellular gene and relies almost entirely on the host-cell
machinery for gene expression. Although this strategy accommodates a
viral genome that encodes a limited number of protein products, it
necessitates that the genome also contain a large array of cis-acting
elements that direct the host-cell machinery to function in viral gene
expression. Most of these elements lie in the long terminal repeats
(LTRs) of the proviral DNA (Fig. 1). The provirus is significantly
longer than the viral genomic RNA in both the 5 and 3 directions. The
U3 region that is found upstream of the transcription start site contains
the majority of cis-acting control elements that regulate transcriptional
initiation by cellular RNA polymerase II. The U3, R, and U5 regions of
the 3 LTR contain the cis-acting control elements involved in
posttranscriptional processing of the 3 end of the RNA product.
Contents
Transcription
Processing of Retroviral RNA
Effects of Proviral Integration on Host Gene Expression
Concluding Remarks
References
Figures
Figure 1