Replication of Viruses

 The pathological effects of the

diseases caused by viruses result
from the interplay of several
factors:-

 Toxic effects of viral gene products

on the metabolism of infected cells.

 Reactions of the host to infected

cells expressing viral genes

 Modification of host gene

expression by structural or
functional interactions with the
 Host Range, Susceptibility,
and Permissiveness
 The process of infection begins with the
coming together of a virus particle and a
susceptible host cell.

 The host range of a virus defines both the

kinds of tissue cells and animal species
that it can infect and in which it can
multiply (wide Vs narrow).

 Susceptibility defines the capacity of a

cell or an animal to become infected.
 Relevant to understanding of
viral pathogenesis at the
molecular level.

 Important in the area of antiviral

chemotherapy where it is
needed to determine what
stages are likely to be potential
targets or susceptible to
chemotherapeutic agents.
 To infect a cell, the virion must attach to
the cell surface, penetrate the cell, and
become sufficiently uncoated to make its
genome accessible to viral or host
machinery for transcription or translation.

 The cell acts as a factory providing the

substrates, energy, and machinery
necessary for synthesis of viral proteins
and replication of the genome.

 Each infected cell may produce as many as

105 particles(burst size), only 1-10% of
which are infectious
 Types of Infection
►Infection of a cell may be:-

► Productive (permissive).

► Abortive (non-permissive, defective).

► Stringent or restrictive(transient
permissiveness).

► Transforming.
► Virusreplication can be divided into eight
stages, namely: Attachment, penetration,
uncoating, genome replication, gene
expression, assembly, maturation and
release.

► These are purely arbitrary divisions, used

here for convenience in explaining the
replication cycle of a non- existing “
typical “ virus.

► Notat all stages described here are

detectable as distinct stages for all
viruses, often they “blur” together and
These stages can be classified into three
phases:-

I – Initiation phase
- Attachment
- Penetration
- Uncoating
II - Replication phase
- DNA Synthesis
- RNA Synthesis
- Protein synthesis
III - Release phase
- Assembly
- Maturation
- Exit from cell
Latent Period
 Attachment (Adsorption)

 Attachment constitutes the specific

binding of a viral protein VAP to a
constituent of the cell surface (receptor/
anti-receptor).

 Complex viruses may have more than one

species of antireceptor molecules.

 Anti-receptor molecules may have several

domains, each of which may react with a
different receptor.
 Mutations in the genes specifying
anti-receptors may cause loss of the
capacity to interact with certain
receptors.

 Receptors identified thus far are

largely glycoproteins or glycolipids.

 Repulsion between virus and cell

membrane impedes attachment
because both are negatively
charged.
 Attachment requires ions to reduce
electrostatic repulsion, but it is
largely independent of temperature
and energy.

 Attachment results from random

collision between virions and cell
surface at a frequency of 10-3 to 10-4
leading to a physical complementary
union.

 Early binding is reversible and firm

binding requires specific receptor
anti- receptor interaction.
 The susceptibility of a cell is limited
by the availability of appropriate
receptors, and not all cells in an
otherwise susceptible organism
express receptors.

 Attachment of viruses to cells in

many instances leads to irreversible
changes in the structure of the
virion.

 In some instances, however, when

penetration does not ensue, the
virus can detach and elute from cell
 Some viruses have specific
mechanisms for detachment
(neuraminidase).

 Elution leads to changes in the virus

VAP which decrease or eliminate the
possibility of subsequent attachment
to other cells.
 Penetration
►An energy dependent step that occurs
almost instantaneously after
attachment and it involves one of three
mechanisms:

►Endocytosis ( viriopexis )of the virus

particle resulting in accumulation of virus
particles inside cytoplasmic vesicles. Most
common.

►Fusion of the virion envelope with the

cellular membrane (Requires fusion
►Translocation of the entire virus
across the plasma membrane.
Rare and poorly understood.

►Penetration may be pH
independent and it is usually
immediately followed
(inseparable) by uncoating.
 Uncoating
► It is poorly understood.

► The virus capsid is completely or partially

removed and the virus genome exposed,
usually in the form of a nucleoprotein
complex.

► Uncoating may be initiated by attachment

to the receptor or prompted by the acidic
environment or proterases found in an
endosome or lysosome.
 Expression and Replication of
viral Genomes
►DNA Viruses
 All DNA viruses, except poxviurses, replicate in
the nucleus.

 They utilize cellular RNA polymerase ( DNA –

dependent RNA Polymerase) for transcription.

 Simple DNA viruses ( Parvo and Papovaviruses )

utilize host cell DNA – dependent DNA
polymerase, whereas the larger more complex
ones ( adeno, herpes, and poxviruses) encode
their own polymerases.
 Viral polymerases are faster but less
precise than cell polymerase causing a
higher mutation rate and providing a
target for antiviral drugs.

 In a few instances it is cellular enzymes

that replicate the viral genome assisted
by viral proteins ( parvovirus).

 In most cases the opposite is true, viral

enzymes are responsible for genome
replication although they utilize cellular
proteins to aid this.
 All DNA viruses known to infect
vertebrates contain a monopartite
genome.

 The fidelity of DNA replication is such that

only one mistake is made in 109 – 1010
base pair replications compared with one
in 103-104 for RNA viruses.

 Error – free replication arises from the

ability of DNA polymerase to proof-read
the DNA which they have just synthesized.

 In contrast, RNA polymerases need not be

self- correcting in as much as relatively
high error rates can be tolerated.
►RNA Viruses
 Most RNA viruses replicate in the cytoplasm
using their own transcriptases, exceptions to this
being influenza and retroviruses, part of the
replicative cycle of which take place in the
nucleus.

 Virion - associated RNA polymerases have the

activities of RNA polymerase, 5' capping, and 3'
polyadenylation.

 Host cells can not replicate nucleic acid in the

cytoplasm, so viruses that replicate in the
cytoplasm carry all enzymes necessary for their
replication and this applies to poxviruses and
most RNA viruses.
 Replication and transcription of RNA
viruses are similar processes as the
template is RNA in both cases, and
ds RNA intermediates are formed.

 Since RNA is degraded relatively

quickly, the RNA polymerase must be
provided or synthesized soon after
uncoating to generate more viral
RNA, or the infection is aborted.
 The genomes of ssRNA viruses are
either:

- Monopartite ( picorna, toga,

paramyxo,
rhabdo, corona, and retroviruses) or

- Multipartite ( orthomyxo, arena, and

bunyaviruses).
►DNA and RNA Viruses
 The virus must be able to interact with the
cell biosynthetic machinery according to
the biochemical rules of the cell.

 Transcription and hence translation

usually proceed in two phases, early and
late.

- The early phase results in the synthesis of

regulatory proteins and enzymes
necessary for replication of viral nucleic
acid.

- The late phase leads to the synthesis of

 Transcription of the viral genes is
regulated by the interaction of specific
DNA – binding proteins with promoter and
enhancer elements in the viral genome.

 Cells from different tissues or species

express different DNA- binding proteins.

 Different DNA and RNA viruses control

the duration, sequence and quantity of
viral gene expression and protein
synthesis in different ways. The more
complex viruses encode their own
transcriptional activators.
 Translation proceeds in essentially
the same fashion as eukaryotic
mRNA utilizing cellular tRNA and
initiation factors.

 Posttranslational modification takes

place utilizing cellular pathways.

 Structural proteins of the virus may

act as repressors of transcription by
binding to viral DNA or RNA.
 Viruses employ different tactics to
promote preferential translation of their
viral mRNA:-

- In many cases the concentration of viral

mRNA
in the cell is so large that it occupies
most of
the ribosomes.

- Block the egress of cellular mRNA from

the nucleus.

- Inhibit cellular macromolecular synthesis

and induce degredation of the cell’s DNA
and mRNA.
Expression and Replication of
viral Genomes
I- RNA Viruses

1- Positive (+) strand RNA viruses coding for one

Genome – sized m RNA ( polio, Flavi, HCV)

► Their coding domains are translated in their entirety.

► The product of translation, the polyprotein, is then
cleaved.
► Synthesis of complementary full- length (-) strand RNA.
► The (-) strand RNA in turn serves as a template to make
more(+) strand RNAs .
 Flow of events during the
replication of Picornaviruses
2- Positive (+) Strand RNA viruses coding for one
or more subgenomic mRNAs (Toga, corona, calici,
HEV).

► Only a portion (the 5' end) of the genomic RNA is

available for translation in the first round of protein
synthesis.

► A (-) strand is then synthesized, and this RNA in turn

serves as a template for two size classes of (+) RNA
molecules.

► Cleavage clearly involves virus- specified poteases,

and the polyprotein itself is enzymatically active in
Trans.

► Two or more subgenomic mRNA species are made in

 Flow of events during the
replication of Togaviruses.
►Central to the replication of (+) strand
viruses is the capability of the genomic
RNA to serve as mRNA after infection.

►The consequences are two fold:

► First, enzymes responsible for the replication of
the genome are made after infection

► Second, because all (+) strand genomes are

monopartite, the initial products of translation
of both genomic RNA and mRNA species are
necessarily a single protein.
 3- Retroviruses
► First step in replication is synthesis of a DNA
strand complementary to the RNA genome,
followed by digestion of RNA by a nuclease
(ribonuclease H in the virion), and finally
synthesis of a complementary DNA strand.

► The linear ds DNA translocated into the

nucleus integrates into the host genome
(Provirus).

► The products of transcription are genome-

length RNA molecules (efficiently packaged
into virions), and shorter, spliced mRNAs that
are translated to yield polyproteins that are
 Flow of events during the
replication of retroviruses.
 4- Non segmented Negative (-) strand RNA viruses

► They have their transcriptases packaged in the

virion.

► Thetranscription of the viral genome is the first

event after entry into cells (multiple functional
mRNAs are produced).

► Replicationbegins under the direction of newly

synthesized viral proteins, a full-length(+)
strand is made and serves as a template for the
synthesis of (-) strand genomic RNA
5- Segmented Negative strand RNA viruses

► The first step involves the synthesis of mRNAs

from each segment of the genomic RNA.

► The mRNAs of influenza virus have

heterogeneous nonviral 5’ end sequences (8 –
18 nucleotides ) that are stolen” from the host
cell mRNA molecules by viral proteins.

► The newly synthesized viral proteins replicate

the genomic RNA segments to yield precise (+)
strand copies of the virion RNAs

► A unique characteristic of them is reassortment

of their genes in cells infected by more than
one virion of the same group introducing new
Flow of events during the replication
of Orthomyxoviruses and
Paramyxoviruses.
►The genes of (-) strand viruses serves as
template for transcription only.

►The consequences are three- fold:-

► First, the virus must bring into the infected cell the
transcriptase to make its mRNAs.

► Second, naked RNA extracted from virions is not

infectious .

► Third, mRNAs produced are gene unit length, they

specify a single polypeptide.

►Consequently, the (+) transcript which functions as

mRNA is different form the (+) strand RNA which serves
as the template for progeny virus even though both are
 6- Ambisenes RNA Viruses
(Arenaviruses and Bunyaviruses)

► The expression of this information takes place

in two stages.

► The genomic RNA is transcribed to yield (+) strand

subgenomic size mRNA.

► The appropriate full size complementary RNA is then

transcribed to yield subgenomic size mRNA.

► Because the replicative cycles begin with the

transcription of genomic RNA, the ambisense
viruses must carry their own polymerase into
the infected cell.
 7- Double Stranded RNA viurses

► The multipartite reovirus genome is transcribed within

the partially opened capsid by a polymerase packaged
into the virion

► The 10 mRNA (+) strand species are extruded from the

exposed vertices of the capsid.

► The mRNA molecules have two functions:

► first, they are translated as monocistronic messages to

yield the viral proteins.

► Second, one RNA of each of the 10 species assemble

within a precursor of particle in which it servers as a
template for synthesis of the complementary strand,
yielding ds genome segments.
Flow of events during the replication of
Reoviruses
►II- DNA Viruses
1- Double – Stranded DNA Viruses that Replicate

in the Nucleus

► Significant differences exist in the replication strategies

of Nuclear viruses.

► Papovaviruses encode a single protein that binds in

close proximity to the origin of viral DNA synthesis,
stimulates the cellular polymerase complex to replicate
the viral DNA, and acts as a helicase.

► Adenoviruses encode a DNA polymerase but depend on

the host cells for all other functions involved in the
synthesis of their DNA.

► At The other extreme are the herpesviruses; HSV

encodes numerous proteins involved in the pathway of
the synthesis of DNA .
Flow of events during the replication
of herpesviruses (herpes simplex
viruses).
 2- Double stranded DNA Viruses that replicate in

the cytoplasm

► Transcriptional events and most of the other

events in the reproductive cycle seem to take
place in the cytoplasm.

► Poxviurses have evolved all of the factors

necessary for transcription and replication of
their genome.

► Because host transcriptional factors are not

involved, the cis - acting sites for the synthesis
and processing of the mRNA have diverged
from those of the host.

► The initial transcription occurs in the core of

the virion, the protein products of these
 3- Single- stranded DNA viruses ( Parvoviruses)

► Multiplication requires the synthesis of a DNA

strand complementary to the ss gnomic DNA in
the nucleus and transcription of the genome.

► The B19 virus replicates in mitotically active

cells and prefers cells of the erythroid lineage.

► Factors available only during the S phase of the

cell’s growth cycle and cellular DNA polymerase
are required to generate a complementary DNA
strand.

► A ds DNA version of the virion genome is

required for transcription and replication.
► Inverted repeat sequences of DNA at both ends
of the genome facilitate viral DNA synthesis. It
forms a ds molecule in the form of hairpin loops.

► The palindromic sequence (about 115 bases at

both ends) can fold back on it self and forms ds
sequences stabilized by hydrogen bonding in the
form of hairpin Y or T shape.

► The ds DNA replicative intermediate is

transcribed by cellular RNA polymerases and
replicated by DNA polymerase.

► In the absence of a helper virus, the genomes of

dependent parvovirus appear to integrate into a
specific locus on a human chromosome
Flow of events during the replication
of Parvoviruses
 4- Hepadnaviruses

► Hepadnaviruses have a circular partially

ds DNA genome. They replicate in the
nucleus.

► The gap in the DNA of the virus is

repaired first by a DNA polymerase
packaged into virion.

► the genome is then transcribed into two

classes of RNA molecules; mRNAs
specifying proteins and a full length RNA
that serves as a template for the
synthesis of genomic DNA by a virally
 Flow of events during the replication
of Hepadnaviruses (hepatitis B virus).
 Assembly, Maturation, and Egress of
viruses from infected cells

► Assembly of DNA viruses, except

poxviruses, occurs in the nucleus and
requires transport of the virion proteins
into the nucleus.

► Assembly of pox and RNA viruses takes

place in the cytoplasm.

► The assembly process begins when the

concentration of structural proteins in the
cell is sufficient to thermodynamically
drive the process, much like a
crystalization reaction.
► Structuralproteins of simple icosahedral
viruses can aggregate spontaneously to
from structural units, which in turn
assemble into empty capsids (procasids).

► Somehow, the viral nucleic acid now

enters this structure via a mechanism that
seems to involve a nucleotide sequence
known as the “ packing sequence”.

► Helical
viruses assemble by adding blocks
during coiling of the viral nucleic acid.
► Maturation and release are determined in part
by site of replication and the presence of an
envelope.

► Acquisitionof an envelope occurs after

association of the nucleocapsid with regions of
host cell membrane modified by matrix protein
and glycoproteins.

► Matrixproteins line and promote the adhesion

of nuclecocapsids with the modified membrane.

► Asmore interactions occur, the membrane

surrounds the nucleocapsid and the virus buds
from the membrane .
 strategies for maturation
► Three
fundamental strategies for
maturation have been described:-

I- Intracellular assembly and Maturation

- Nonenveloped viruses cause disintegration

of the
infected cell for their egress.

II- Strategy of enveloped viruses

-The last step in assembly of (-) strand RNA

► Viruses that mature and egress by budding
vary considerably in their effects on host cell
metabolism and integrity.

► They range from highly cytolytic (toga,

paramyxo) to viruses which are frequently
noncytolytic
(retroviruses) .

► By virtue of the viral glycoprotein insertion

into the cell surface, however, these viruses
import upon the cell a new antigenic
specificity and the infected cell can and does
become a target for the immune mechanisms
III- Strategy for Herpesviruses

- They
assemble their nucleocapsid in the
nucleus.

- Envelopment and maturation occur at

the inner lamella of the nuclear
membrane

- Herpesvirurses are cytolytic and

invariably destroy the cell in which they

multiply.

- They also import new antigens on the

 Glycosylation and Budding

► Inthe glycosylation of their proteins, viruses

use existing pathways.

► This involves a “ signal sequence “ of 15-30

hydrophobic amino acids that facilitate binding
to a receptor on the cytoplasmic side of the
RER.

► Itthen passes through the lipid bilayer to the

luminal side where the signal sequences is
removed by a signal peptidase allowing the
addition of oligosaccharides.
► Glucoseis then removed by glucosidase
(trimming).

► Theviral glycoprotein is then transported to the

Golgi apparatus probably inside a coated
vesicle, where the core carbohydrate is further
modified and acylated (addition of fatty acids).

► Anothercoated vesicle now transports the

acylated glycorprotein to the plasma
membrane, probably with the help of a leading
sequence that finds the destination (postal
address or zip code(.
► Envelope glycoproteins are then cleaved into 2
poly- peptide chains that remain covalently
bound by S-S bonds.

► Thenthe hydrophilic N-terminus of the

glycoprotein finds itself projecting from the
external surface of the membrane while the
hydropobic domain near the c-terminus remains
anchored in the lipid bilayer.

► Budding is a form of exocytosis (reversed

endocytosis) and viruses remain cell-
associated for few hours and large numbers of
viruses are released in consecutive waves.
 Variability in viral Genomes and viral
Multiplication

► On passage, viruses tend to yield defective

mutants.

► It is convenient to classify defective viruses

into two groups.

Viruses in the first group lack one or more

essential genes and therefore are incapable of
independent replication without a helper virus.

- They can transform infected cells or

transactivate oncogenic viruses in causing the
 The second group comprises viruses which contain
mutations and deletions and therefore can not replicate
mutations and deletions and therefore can not replicate
in an efficient fashion.

- Chronic debilitating infections of the CNS might in some

fashion be related to viruses that are sluggish in their
replication, in their ability to destroy the infected cells,
or in their ability to alter the infected cell sufficiently to
make it a target for the immune system of the host..

- Genetically engineered viruses lacking one or several

genes and which might be classified as defective may
ultimately be viruses greatest gift to mankind; the
means for the introduction of genes to complement
genetic deficits or to selectively destroy cancer cells.