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VIRAL REPLICATION

Viral replication is the term used by virologists to describe the formation of biological viruses
during the infection process in the target host cells. Viruses must first get into the cell
before viral replication can occur. From the perspective of the virus, the purpose of viral
replication is to allow production and survival of its kind. By generating abundant copies
of its genome and packaging these copies into viruses, the virus is able to continue
infecting new hosts. Replication between viruses is greatly varied and depends on the
type of genes involved. Viral populations do not grow through cell division, because they
are acellular; instead, they use the machinery and metabolism of a host cell to produce
multiple copies of themselves, and they assemble in the cell.
The life cycle of viruses differs greatly between species but there are six basic stages in the life
cycle of viruses:

Attachment is a specific binding between viral capsid proteins and specific receptors on
the host cellular surface. This specificity determines the host range of a virus. For
example, HIV infects only human T cells, because its surface protein, gp120, can interact
with CD4 and receptors on the T cell's surface. This mechanism has evolved to favour
those viruses that only infect cells in which they are capable of replication. Attachment to
the receptor can induce the viral-envelope protein to undergo changes that results in the
fusion of viral and cellular membranes.
Penetration follows attachment; viruses enter the host cell through receptor mediated
endocytosis or membrane fusion. This is often called viral entry. The infection of plant
cells is different from that of animal cells. Plants have a rigid cell wall made of cellulose
and viruses can only get inside the cells after trauma to the cell wall. Viruses such as
tobacco mosaic virus can also move directly in plants, from cell to cell, through pores
called plasmodesmata.[83] Bacteria, like plants, have strong cell walls that a virus must
breach to infect the cell. Some viruses have evolved mechanisms that inject their genome
into the bacterial cell while the viral capsid remains outside.

Uncoating is a process in which the viral capsid is degraded by viral enzymes or host
enzymes thus releasing the viral genomic nucleic acid.

Replication involves synthesis of viral messenger RNA (mRNA) for viruses except
positive sense RNA viruses (see above), viral protein synthesis and assembly of viral
proteins and viral genome replication.

Following the assembly of the virus particles, post-translational modification of the viral
proteins often occurs. In viruses such as HIV, this modification (sometimes called
maturation) occurs after the virus has been released from the host cell.

Viruses are released from the host cell by lysisa process that kills the cell by bursting
its membrane. Enveloped viruses (e.g., HIV) typically are released from the host cell by
budding. During this process the virus acquires its envelope, which is a modified piece of
the host's plasma membrane.

SMB 301:
Lecturer:

INTRO TO VIROLOGY
KU-MAIN CAMPUS
Shem Peter Mutua Mutuiri smutuiri@gmail.com

BSc. MICROBIOLOGY

Protein synthesis
Proteins are essential to life. Cells produce new protein molecules from amino acid
building blocks based on information coded in DNA. Each type of protein is a specialist
that only performs one function, so if a cell needs to do something new, it must make a
new protein. Viruses force the cell to make new proteins that the cell does not need, but
are needed for the virus to reproduce. Protein synthesis basically consists of two major
steps: transcription and translation.
Transcription is the process where information in DNA, called the genetic code, is used
to produce RNA copies called messenger RNA (mRNA). These migrate through the cell
and carry the code to ribosomes where it is used to make proteins. This is called
translation because the protein's amino acid structure is determined by the mRNA's code.
Some RNA genes of viruses function directly as mRNA without further modification. For
this reason, these viruses are called positive-sense RNA viruses. In other RNA viruses,
the RNA is a complementary copy of mRNA and these viruses rely on the cell's or their
own enzyme to make mRNA. These are called negative-sense RNA viruses. In viruses
made from DNA, the method of mRNA production is similar to that of the cell. The
species of viruses called retroviruses behave completely differently: they have RNA, but
inside the host cell a DNA copy of their RNA is made. This DNA is then incorporated
into the host's, and copied into mRNA by the cell's normal pathways.

The genetic material within viruses, and the method by which the material is replicated, vary
between different types of viruses.
DNA viruses
The genome replication of most DNA viruses takes place in the cell's nucleus. If the cell
has the appropriate receptor on its surface, these viruses enter the cell by fusion with the
cell membrane or by endocytosis. Most DNA viruses are entirely dependent on the host
cell's DNA and RNA synthesising machinery, and RNA processing machinery. The viral
genome must cross the cell's nuclear membrane to access this machinery.
RNA viruses
These viruses are unique because their genetic information is encoded in RNA.
Replication usually takes place in the cytoplasm. RNA viruses can be placed into about
four different groups depending on their modes of replication. The polarity (whether or
not it can be used directly to make proteins) of the RNA largely determines the replicative
mechanism, and whether the genetic material is single-stranded or double-stranded. RNA
viruses use their own RNA replicase enzymes to create copies of their genomes.
Reverse transcribing viruses
These replicate using reverse transcription, which is the formation of DNA from an RNA
template. Reverse transcribing viruses containing RNA genomes use a DNA intermediate
to replicate, whereas those containing DNA genomes use an RNA intermediate during
genome replication. Both types use the reverse transcriptase enzyme to carry out the
nucleic acid conversion. Retroviruses often integrate the DNA produced by reverse
transcription into the host genome. They are susceptible to antiviral drugs that inhibit the
reverse transcriptase enzyme, e.g. zidovudine and lamivudine. An example of the first
type is HIV, which is a retrovirus. Examples of the second type are the Hepadnaviridae,
which includes Hepatitis B virus.
SMB 301:
Lecturer:

INTRO TO VIROLOGY
KU-MAIN CAMPUS
Shem Peter Mutua Mutuiri smutuiri@gmail.com

BSc. MICROBIOLOGY

Baltimore Classification System


Viruses are classed into 7 types of genes, each of which have their own families of viruses,
which in turn have differing replication strategies themselves. David Baltimore, a Nobel Prizewinning biologist, devised a system called the Baltimore Classification System to classify
different viruses based on their unique replication strategy. There are seven different replication
strategies based on this system (Baltimore Class I, II, III, IV, V, VI, VII). The seven classes of
viruses are listed here briefly and in generalities.

Class 1: Double stranded DNA viruses


This type of virus usually must enter the host nucleus before it is able to replicate. Furthermore,
these viruses require host cell polymerases to replicate its genome and hence is highly dependent
on the cell cycle. Proper infection and production of progeny requires that the cell be in
replication as that is when the cell's polymerases are active. The virus may induce the cell to
forcefully undergo cell division, and chronically, this may lead to transformation of the cell and
ultimately, cancer. An example of a family within this classification is the Adenoviridae.
Adenovirus genome consists of a linear double stranded DNA of about 36 kilobases pair;
attached in a covalent linkage to 5 terminus of the DNA. This is a protein component essential
for infectivity of DNA.Replication of viral DNA occurs in the nucleus.After the viral particle has
been transported to the nucleus, the core is released and converted to DNA-Histone compex.
Early transcription is done by RNA polymerase of the host and a number of primary transcripts
are made.The transcripts are spliced, capped and equally polyadenylated to give several different
mRNAs. Early proteins are involved in the regulation of DNA replication, later proteins are the
virus coat proteins. Viral DNA replication uses viral encoded proteins as a primer and another
virus encoded protein as DNA polymerase. Replication of a linear dsDNA molecule e.g.
adenovins, the imitation can begin at either end or at both ends simultaneously.Two strands are
produced which are rounded/double single strands. They later cyclize by means of the inverted
terminal repeats and a new complimentary strand is synthesized beginning from the 5 end. The
product being another double stranded molecule. This mechanism of replication is interesting
because it does not involve the formation of discontinuous fragments of DNA of the lagging
strand as occurs in conventional DNA replication.
There is only one well studied example in which a class 1 virus is not replicating within the
nucleus, that is the Poxvirus family, a highly pathogenic virus that infects vertebrates. The
example is a smallpox virus.

SMB 301:
Lecturer:

INTRO TO VIROLOGY
KU-MAIN CAMPUS
Shem Peter Mutua Mutuiri smutuiri@gmail.com

BSc. MICROBIOLOGY

Class 2: Single stranded DNA viruses


Viruses that fall under this category includes ones that are not as well studied, but still do pertain
highly to vertebrates. Two examples include the Circoviridae and Parvoviridae. They replicate
within the nucleus, and form a double stranded DNA intermediate during replication. A human
Circovirus called TTV is included within this classification and is found in most all humans,
infecting them asymptomatically in nearly every major organ.
Class 3: Double stranded RNA viruses
Like most viruses with RNA genomes, double stranded RNA viruses do not rely on host
polymerases for replication to extent that viruses with DNA genomes do. Double stranded RNA
viruses are not as well studied as other classes. This class includes two major families, the
Reoviridae and Birnaviridae. Replication is monocistronic and includes individual, segmented
genomes, meaning that each of the genes code for only one protein, unlike other viruses which
exhibit more complex translation.
Class 4 & 5: Single stranded RNA viruses
These viruses consist of two types, however both share the fact that replication is primarily in the
cytoplasm, and that replication is not as dependent on the cell cycle as DNA viruses. This class
of viruses are also one of the most studied types of viruses, alongside the double stranded DNA
viruses.
Class 4: Single stranded RNA viruses - Positive (+) sense
The positive sense RNA viruses and indeed all genes defined as positive sense can be directly
accessed by host ribosomes to immediately form proteins. These can be divided into two groups,
both of which reproduce in the cytoplasm:

Viruses with polycistronic mRNA where the genome RNA forms the mRNA and is
translated into a polyprotein product that is subsequently cleaved to form the mature
proteins. This means that the gene can utilize a few methods in which to produce proteins
from the same strand of RNA, all in the sake of reducing the size of its gene.
Viruses with complex transcription, for which subgenomic mRNAs, ribosomal
frameshifting and proteolytic processing of polyproteins may be used. All of which are
different mechanisms with which to produce proteins from the same strand of RNA.

Examples of this class include the families Coronaviridae, Flaviviridae and Picornaviridae.
Class 5: Single stranded RNA viruses - Negative (-) sense
The negative sense RNA viruses and indeed all genes defined as negative sense cannot be
directly accessed by host polymerases to immediately form proteins. Instead, they must be

SMB 301:
Lecturer:

INTRO TO VIROLOGY
KU-MAIN CAMPUS
Shem Peter Mutua Mutuiri smutuiri@gmail.com

BSc. MICROBIOLOGY

transcribed by viral polymerases into a "readable" form, which is the positive sense reciprocal.
These can also be divided into two groups:

Viruses containing non segmented genomes for which the first step in replication is
transcription from the (-) stranded genome by the viral RNA-dependent RNA polymerase
to yield monocistronic mRNAs that code for the various viral proteins. A (+) sense
genome copy is then produced that serves as template for production of the (-) strand
genome. Replication is within the cytoplasm.
Viruses with segmented genomes for which replication occurs in the nucleus and for
which the viral RNA-dependent RNA polymerase produces monocistronic mRNAs from
each genome segment. The largest difference between the two is the location of
replication.

Examples in this class include the families Orthomyxoviridae, Paramyxoviridae, Bunyaviridae,


Filoviridae and Rhabdoviridae (which includes rabies).
The negative RNA is transcribed in the cytoplasm of the cell into distinct RNA namely;
1.+ssRNA which acts as mRNA.
2.+ssRNA which is complimentary of the viral genome.
The +ssRNA is used as a template for the synthesis of -ssRNA( viral genome) mRNA may be
monocistronic coding for only one protein.The poly A-tail is not at the end of mRNA and serves
as stop codon.The synthesis of RNA polymerase which initiates the formation of many +ss
sRNA is also in place. The transcription of viral mRNA leads to synthesis of coat protein and
copying the full length +ssRNA leads to the formation of full length ssRNA, Assembly follows,
where the nucleocapsid proteins and the capsid envelope proteins are brought together. The
nucleic acid(RNA) of the orthomyxovirus exists in different fragments in the capsid i.e. exists a
segmented genome e.g. influenza virus has 8 fragments or segments of RNA in the capsid.
Class 6: Positive (+) sense single stranded RNA viruses that replicate through a DNA
intermediate
A well studied family of this class of viruses include the retroviruses. One defining feature is the
use of reverse transcriptase to convert the positive sense RNA into DNA. Instead of using the
RNA for templates of proteins, they use DNA to create the templates, which is spliced into the
host genome using integrase. Replication can then commence with the help of the host cell's
polymerases. A well studied example includes HIV.RNA is converted dsDNA which is a linear
molecule by the enzyme reverse transcription in the cytoplasm. Reverse transcription enzyme is
essentially a DNA polymerase with three enzymatic activities.
1.synthesis of DNA with a DNA template.
2.Synthesis of DNA with a RNA template.

SMB 301:
Lecturer:

INTRO TO VIROLOGY
KU-MAIN CAMPUS
Shem Peter Mutua Mutuiri smutuiri@gmail.com

BSc. MICROBIOLOGY

3.Ribonuclease-H activity (it degrades the RNA strand of a RNA-DNA hybrid)


A primer is required by the two enzymes,reverse tanscription needs a primer for DNA synthesis
which is a specific cellular transfer RNA (+RNA). Using the RNA primer the 100 or so nucleoids
at the 5 terminus of the RNA are reversed transcribed into DNA. Once transcription reaches the
5end of RNA, the transcription process stops. The ribonuclease H activity leads to formation of
a small, single stranded DNA that is complimentary to the RNA segment at the other end of the
viral DNA. This small ssDNA hybrid with the other end of the viral RNA molecule,while the
copying molecule of the viral RNA sequence is continued. The continued action of the reverse
transcriptase and ribonuclease H leads to formation of dsDNA molecule with long terminal
repeats at each end. The lts contain strong promoters of transcription and are involved in the
integration process. A provirus is a stable genetic element after a succesful intergration of the
viral DNA into the host DNA.
Class 7: Double stranded DNA viruses that replicate through a single stranded RNA
intermediate
This small group of viruses, exemplified by the Hepatitis B virus, have a double-stranded,
gapped genome that is subsequently filled in to form a covalently closed circle (ccc DNA) that
serves as a template for production of viral mRNAs and a subgenomic RNA. The pregenome
RNA serves as template for the viral reverse transcriptase and for production of the DNA
genome.

SMB 301:
Lecturer:

INTRO TO VIROLOGY
KU-MAIN CAMPUS
Shem Peter Mutua Mutuiri smutuiri@gmail.com

BSc. MICROBIOLOGY

SUMMARY

I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc)


II: Single-stranded (+)sense DNA (Parvoviruses)
III: Double-stranded RNA (Reoviruses; Birnaviruses)
IV: Single-stranded (+)sense RNA (Picornaviruses; Togaviruses, etc)
V: Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses, etc)
VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle (Retroviruses)
VII: Double-stranded DNA with RNA intermediate (Hepadnaviruses)

REFERENCE: 88I.N.J. Dimmock et al. "Introduction to Modern Virology, 6th edition."


Blackwell Publishing, 2007.
SMB 301:
Lecturer:

INTRO TO VIROLOGY
KU-MAIN CAMPUS
Shem Peter Mutua Mutuiri smutuiri@gmail.com

BSc. MICROBIOLOGY