Replication of VirusesReplication of Viruses

The pathological effects of the The pathological effects of the diseases caused by viruses result diseases caused by viruses result from the interplay of several from the interplay of several factorsfactors::

Toxic effects of viral gene products Toxic effects of viral gene products on the metabolism of infected cellson the metabolism of infected cells..

Reactions of the host to infected Reactions of the host to infected cells expressing viral genescells expressing viral genes

Modification of host gene Modification of host gene expression by structural or expression by structural or functionalfunctional interactions with the interactions with the genetic material of the virusgenetic material of the virus..

HostHost Range, Susceptibility, Range, Susceptibility, and Permissivenessand Permissiveness

The process of infection begins with the The process of infection begins with the coming together of a virus particle and a coming together of a virus particle and a susceptible host cellsusceptible host cell..

The host range of a virus defines both the The host range of a virus defines both the kinds of tissue cells and animal species kinds of tissue cells and animal species that it can infect and in which it can that it can infect and in which it can multiply (wide Vs narrow).multiply (wide Vs narrow).

Susceptibility defines the capacity of a cell Susceptibility defines the capacity of a cell or an animal to become infected.or an animal to become infected.

Viral Replication: Basic Viral Replication: Basic ConceptsConcepts

►Viruses are obligate intracellular parasitesViruses are obligate intracellular parasites

►Viruses carry their genome (RNA or DNA) Viruses carry their genome (RNA or DNA) and sometimes functional proteins and sometimes functional proteins required for early steps in replication required for early steps in replication cyclecycle

►Viruses depend on host cell machinery to Viruses depend on host cell machinery to complete replication cycle and must complete replication cycle and must commandeer that machinery to commandeer that machinery to successfully replicatesuccessfully replicate

Viral Replication: Basic Viral Replication: Basic ConceptsConcepts

►Replication cycle producesReplication cycle produces - - Functional RNA’s and proteinsFunctional RNA’s and proteins - - Genomic RNA or DNA and structural Genomic RNA or DNA and structural

proteinsproteins

►Up to 100.000 new particles are produced Up to 100.000 new particles are produced by each cycleby each cycle

- - Referred to as burst sizeReferred to as burst size - - Many are defectiveMany are defective - - End of ‘eclipse’ phaseEnd of ‘eclipse’ phase

►Replication may be cytolytic or non-Replication may be cytolytic or non-cytolyticcytolytic

Latent Period

Viral replication is a complex process that Viral replication is a complex process that involves multiple interactions at the involves multiple interactions at the molecular level. molecular level.

Discussion will concentrate on aspects Discussion will concentrate on aspects relevant to understanding of viral relevant to understanding of viral pathogenesis at the molecular level.pathogenesis at the molecular level.

Important in the area of antiviral Important in the area of antiviral

chemotherapy where it is needed to chemotherapy where it is needed to determine what stages are likely to be determine what stages are likely to be potential targets or susceptible to potential targets or susceptible to chemotherapeutic agentschemotherapeutic agents..

To infect a cell, the virion must attach to To infect a cell, the virion must attach to the cell surface, penetrate the cell, and the cell surface, penetrate the cell, and become sufficiently uncoated to make its become sufficiently uncoated to make its genome accessible to viral or host genome accessible to viral or host machinery for transcription or translationmachinery for transcription or translation..

The cell acts as a factory providing the The cell acts as a factory providing the substrates, energy, and machinery substrates, energy, and machinery necessary for synthesis of viral proteins necessary for synthesis of viral proteins and replication of the genomeand replication of the genome..

Each infected cell may produce as many Each infected cell may produce as many as 10as 1055 particles(burst size), only 1-10% of particles(burst size), only 1-10% of which are infectious which are infectious

Types of InfectionTypes of Infection►Infection of a cell may be:-Infection of a cell may be:-

► ProductiveProductive (permissive). (permissive).

►AbortiveAbortive (non-permissive, defective). (non-permissive, defective).

► StringentStringent or or restrictiverestrictive(transient (transient

permissiveness).permissiveness).

► Transforming.Transforming.

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

► These are purely arbitrary divisions, used These are purely arbitrary divisions, used here for convenience in explaining the here for convenience in explaining the replication cycle of a non- existing “ typical replication cycle of a non- existing “ typical “ virus. “ virus.

►Not all stages described here are Not all stages described here are detectable as distinct stages for all viruses, detectable as distinct stages for all viruses, often they “blur” together and appear to often they “blur” together and appear to occur almost simultaneouslyoccur almost simultaneously. .

These stages can be divided into three These stages can be divided into three phasesphases::

II – – Initiation phaseInitiation phase- - AttachmentAttachment- - PenetrationPenetration- - UncoatingUncoating

IIII - - Replication phaseReplication phase- - DNA SynthesisDNA Synthesis - - RNA SynthesisRNA Synthesis - - Protein synthesisProtein synthesis

IIIIII - - Release phaseRelease phase- - AssemblyAssembly- - MaturationMaturation- - Exit from cellExit from cell

Steps in Viral Replication: Steps in Viral Replication: AttachmentAttachment

► First StepFirst Step

► Surface protein on virus attaches to specific Surface protein on virus attaches to specific receptor(s) on cell surfacereceptor(s) on cell surface

- - May be specialized proteins with limited May be specialized proteins with limited tissue distribution or more widely tissue distribution or more widely

distributeddistributed - - Virus specific receptor is necessary but not Virus specific receptor is necessary but not

sufficient for viruses to infect cells and sufficient for viruses to infect cells and complete replicative cyclecomplete replicative cycle

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

Complex viruses may have more than one Complex viruses may have more than one

species of antireceptor molecules. species of antireceptor molecules.

Anti-receptor molecules may have several Anti-receptor molecules may have several domains, each of which may react with a domains, each of which may react with a different receptor. different receptor.

Mutations in the genes specifying anti-Mutations in the genes specifying anti-receptors may cause loss of the capacity to receptors may cause loss of the capacity to interact with certain receptors.interact with certain receptors.

Receptors identified thus far are largely glycoproteins Receptors identified thus far are largely glycoproteins or glycolipidsor glycolipids..

Repulsion between virus and cell membrane impedes Repulsion between virus and cell membrane impedes attachment because both are negatively charged.attachment because both are negatively charged.

Attachment, therefore, requires ions to reduce Attachment, therefore, requires ions to reduce electrostatic repulsion, but it is largely independent electrostatic repulsion, but it is largely independent of temperature and energy.of temperature and energy.

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

Early binding is reversible and firm Early binding is reversible and firm binding requires specific receptor binding requires specific receptor anti- receptor interactionanti- receptor interaction..

The susceptibility of a cell is limited The susceptibility of a cell is limited by the availability of appropriate by the availability of appropriate receptors, and not all cells in an receptors, and not all cells in an otherwise susceptible organism otherwise susceptible organism express receptors. express receptors.

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

In some instances, however, when In some instances, however, when penetration does not ensue, the penetration does not ensue, the virus can detach and elute from cell virus can detach and elute from cell surface. surface.

Some viruses have specific Some viruses have specific mechanisms for detachment mechanisms for detachment (neuraminidase). (neuraminidase).

Elution leads to changes in the virus Elution leads to changes in the virus VAP which decrease or eliminate the VAP which decrease or eliminate the possibility of subsequent attachment possibility of subsequent attachment to other cellsto other cells..

PenetrationPenetration► Second StepSecond Step

►An energy dependent step that occurs An energy dependent step that occurs almost instantaneously after attachment almost instantaneously after attachment and it involves one of three mechanismsand it involves one of three mechanisms::

► Endocytosis (viriopexis ) Endocytosis (viriopexis ) of the virus particle of the virus particle resulting in accumulation of virus particles resulting in accumulation of virus particles inside cytoplasmic vesicles. Most common.inside cytoplasmic vesicles. Most common.

► Fusion of the virion envelope with the Fusion of the virion envelope with the cellular membrane cellular membrane (Requires fusion protein in (Requires fusion protein in viral envelope)viral envelope)

►TranslocationTranslocation of the entire virus of the entire virus across the plasma membrane.across the plasma membrane. Rare and poorly understood.Rare and poorly understood.

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

Penetration - EndocytosisPenetration - Endocytosis Most commonly, viruses enter cells by endocytosis (engulfment by the invagination of a section of plasma membrane).

Virus particles accumulate in cytoplasmic vesicles and are subsequenty uncoated.

Endocytosis of Non-enveloped Endocytosis of Non-enveloped RNA VirusesRNA Viruses

►pH dependentpH dependent

- - Cell Receptor (IgG super family)Cell Receptor (IgG super family)

- At low pH, virus becomes lipophilic and - At low pH, virus becomes lipophilic and

forms a pore in the cell membraneforms a pore in the cell membrane

- RNA genome is then ejected through the - RNA genome is then ejected through the

hydrophobic porehydrophobic pore

►Can be inhibited by use of weak bases Can be inhibited by use of weak bases such as ammonium chloride & chloroquinesuch as ammonium chloride & chloroquine

Endocytosis of Enveloped RNA Endocytosis of Enveloped RNA VirusesViruses

Influenza Virus as an example

Endosome is acidic (pH 5-6)

Exposed hydrophobic fusion domain

Differ from nonenveloped viruses in that their envelopes fuse with the membranes of the endosomes

Penetration - FusionPenetration - Fusion►Direct FUSION of the virion envelope Direct FUSION of the virion envelope

with the surface membrane of the cells with the surface membrane of the cells may also take place with some virusesmay also take place with some viruses

►Virion envelope glycoproteins with Virion envelope glycoproteins with fusion activity mediate the melding of fusion activity mediate the melding of the two phospholipids bilayers and the two phospholipids bilayers and mixing of the aqueous compartments mixing of the aqueous compartments previously separated by them.previously separated by them.

► In some viruses a specialized In some viruses a specialized glycoprotein is responsible for fusion glycoprotein is responsible for fusion (eg gp41 of HIV)(eg gp41 of HIV)

Penetration - TranslocationPenetration - Translocation►Translocation – Some non-enveloped Translocation – Some non-enveloped

viruses enter by translocation of the viruses enter by translocation of the whole virus particle across the cell whole virus particle across the cell membrane. membrane.

►They are then uncoated in the They are then uncoated in the cytoplasm.cytoplasm.

►It is not understood how intact virus It is not understood how intact virus particles move directly through cell particles move directly through cell membranesmembranes

Steps in Viral Replication: Steps in Viral Replication: UncoatingUncoating

►Third StepThird Step

►Makes viral nucleic acid available for Makes viral nucleic acid available for transcription to permit multiplication transcription to permit multiplication to proceedto proceed

►Mechanism variably understood Mechanism variably understood depending upon the virusdepending upon the virus

UncoatingUncoating►Uncoating usually occurs after penetration.Uncoating usually occurs after penetration.

►Capsid is removed and genome is exposed Capsid is removed and genome is exposed usually as a nucleoprotein complexusually as a nucleoprotein complex

►Process is poorly understood and variableProcess is poorly understood and variable

► In reoviruses, the capsid only ever partially In reoviruses, the capsid only ever partially disintegrates and replication takes place in disintegrates and replication takes place in a structured particle.a structured particle.

► In poxviruses, host factors induce the In poxviruses, host factors induce the disruption of the virus.disruption of the virus.

UncoatingUncoating► The release of DNA from the core depends The release of DNA from the core depends

upon viral factors made after entry.upon viral factors made after entry.

►Orthomyxo, paramyxo and picornavirus all Orthomyxo, paramyxo and picornavirus all lose the protective envelope or capsid lose the protective envelope or capsid upon entry into the cytoplasm. upon entry into the cytoplasm.

► In the influenza virus, the M2 envelope In the influenza virus, the M2 envelope viral protein allow endosomal protons into viral protein allow endosomal protons into the virion particle resulting in its partial the virion particle resulting in its partial dissolution.dissolution.

► In herpesviruses, adenoviruses and In herpesviruses, adenoviruses and papovaviruses, the capsid is eventually papovaviruses, the capsid is eventually routed along the cytoskeleton to nuclear routed along the cytoskeleton to nuclear membranemembrane

Expression and Replication of Expression and Replication of viral Genomesviral Genomes

►DNA VirusesDNA Viruses

All DNA viruses, except poxviruses, replicate in All DNA viruses, except poxviruses, replicate in the nucleus.the nucleus.

They utilize cellular RNA polymerase (DNA They utilize cellular RNA polymerase (DNA ––

dependent RNA Polymerase) for transcription. dependent RNA Polymerase) for transcription.

Simple DNA viruses (Parvo and Papovaviruses) Simple DNA viruses (Parvo and Papovaviruses) utilize host cell DNA utilize host cell DNA –– dependent DNA dependent DNA polymerase, whereas the larger more complex polymerase, whereas the larger more complex ones ( adeno, herpes, and poxviruses) encode ones ( adeno, herpes, and poxviruses) encode their own DNA polymerases. their own DNA polymerases.

Viral polymerases are faster but less precise than cell polymerase causing a higher mutation rate and providing a target for antiviral drugs.

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.

In a few instances it is cellular enzymes In a few instances it is cellular enzymes that that replicate the viral genome replicate the viral genome assisted by viral proteins assisted by viral proteins (parvovirus). (parvovirus).

In most cases the opposite is true, In most cases the opposite is true, viral enzymes are responsible for viral enzymes are responsible for genome replication although they genome replication although they utilize cellular proteins to aid thisutilize cellular proteins to aid this..

All DNA viruses known to infect All DNA viruses known to infect vertebrates contain a monopartite vertebrates contain a monopartite genome.genome.

►RNA VirusesRNA Viruses

Most RNA viruses replicate in the cytoplasm using Most RNA viruses replicate in the cytoplasm using their own transcriptase, exceptions to this being their own transcriptase, exceptions to this being influenza and retroviruses, part of the replicative influenza and retroviruses, part of the replicative cycle of which take place in the nucleuscycle of which take place in the nucleus..

Virion - associated RNA polymerases have the Virion - associated RNA polymerases have the activities of RNA polymerase, 5' capping, and 3' activities of RNA polymerase, 5' capping, and 3' polyadenylationpolyadenylation..

Host cells cannot replicate nucleic acid in the Host cells cannot replicate nucleic acid in the cytoplasm, so viruses that replicate in the cytoplasm, so viruses that replicate in the cytoplasm carry all enzymes necessary for their cytoplasm carry all enzymes necessary for their replication and this applies to poxviruses and replication and this applies to poxviruses and most RNA viruses. most RNA viruses.

Replication and transcription of RNA Replication and transcription of RNA viruses are similar processes as the viruses are similar processes as the template is RNA in both cases, and template is RNA in both cases, and ds RNA intermediates are formedds RNA intermediates are formed..

Since RNA is degraded relatively Since RNA is degraded relatively quickly, the RNA polymerase must be quickly, the RNA polymerase must be provided or synthesized soon after provided or synthesized soon after uncoating to generate more viral uncoating to generate more viral RNA, or the infection is aborted.RNA, or the infection is aborted.

The genomes of ssRNA viruses are The genomes of ssRNA viruses are eithereither::

- Monopartite- Monopartite ( picorna, toga, paramyxo, ( picorna, toga, paramyxo,

rhabdo, corona, and retroviruses) orrhabdo, corona, and retroviruses) or

- Multipartite- Multipartite ( orthomyxo, arena, and ( orthomyxo, arena, and

bunyaviruses).bunyaviruses).

Most RNA genomes are linearMost RNA genomes are linear

►DNA and RNA VirusesDNA and RNA Viruses

The virus must be able to interact with the The virus must be able to interact with the cell biosynthetic machinery according to cell biosynthetic machinery according to the biochemical rules of the cellthe biochemical rules of the cell. .

Transcription and hence translation usually Transcription and hence translation usually proceed in two phases, early and late. proceed in two phases, early and late.

- The early phase results in the synthesis of The early phase results in the synthesis of regulatory proteins and enzymes necessary regulatory proteins and enzymes necessary for replication of viral nucleic acid. for replication of viral nucleic acid.

- The late phase leads to the synthesis of The late phase leads to the synthesis of structural proteins which are usually made structural proteins which are usually made in excessin excess..

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

Cells from different tissues or species Cells from different tissues or species express different DNA- binding proteins.express different DNA- binding proteins.

Different DNADifferent DNA and RNAand RNA viruses control viruses control the duration, sequence and quantity of the duration, sequence and quantity of viral gene expression and protein viral gene expression and protein synthesis in different ways. synthesis in different ways.

The more complex viruses encode their The more complex viruses encode their own transcriptional activatorsown transcriptional activators..

Translation proceeds in essentially Translation proceeds in essentially the same fashion as eukaryotic the same fashion as eukaryotic mRNA utilizing cellular tRNA and mRNA utilizing cellular tRNA and initiation factors.initiation factors.

Posttranslational modification takes Posttranslational modification takes place utilizing cellular pathways.place utilizing cellular pathways.

Structural proteins of the virus may Structural proteins of the virus may act as repressors of transcription by act as repressors of transcription by binding to viral DNA or RNA.binding to viral DNA or RNA.

Viruses employ different tactics to promote Viruses employ different tactics to promote the preferential translation of their viral the preferential translation of their viral mRNA:-mRNA:-

- In many cases, the concentration of viral - In many cases, the concentration of viral mRNA in the cell is so large that it occupies mRNA in the cell is so large that it occupies most of the ribosomes.most of the ribosomes.

- Block the egress of cellular mRNA from the - Block the egress of cellular mRNA from the nucleus.nucleus.

- Inhibit cellular macromolecular synthesis - Inhibit cellular macromolecular synthesis and induce degradation of the cell’s DNA and and induce degradation of the cell’s DNA and mRNA.mRNA.

- Increase the permeability of the cell - Increase the permeability of the cell membranes which decreases the ribosomal membranes which decreases the ribosomal affinity for cellular mRNA.affinity for cellular mRNA.

Expression andExpression and Replication of Replication of viral Genomesviral Genomes

I- RNA VirusesI- RNA Viruses 1- Positive (+) strand RNA viruses coding for 1- Positive (+) strand RNA viruses coding for one Genome – sized mRNA one Genome – sized mRNA (polio, Flavi, HCV)(polio, Flavi, HCV)

► Their coding domains are translated in their Their coding domains are translated in their entirety. entirety.

► The product of translation, the polyprotein, is then The product of translation, the polyprotein, is then cleaved. cleaved.

► Synthesis of complementary full- length (-) strand Synthesis of complementary full- length (-) strand RNA. RNA.

► The (-) strand RNA in turn serves as a template to The (-) strand RNA in turn serves as a template to make more(+) strand RNAs . make more(+) strand RNAs .

Flow of events during the Flow of events during the replication of Picornavirusesreplication of Picornaviruses

2- Positive (+) Strand RNA viruses coding for 2- Positive (+) Strand RNA viruses coding for one or more subgenomic mRNAs (one or more subgenomic mRNAs (Toga, corona, Toga, corona, calici, HEV)calici, HEV)

► Only a portion (the 5' end) of the genomic RNA is Only a portion (the 5' end) of the genomic RNA is available for translation in the first round of protein available for translation in the first round of protein synthesis. synthesis.

► A (-) strand is then synthesized, and this RNA in turn A (-) strand is then synthesized, and this RNA in turn serves as a template for two size classes of (+) RNA serves as a template for two size classes of (+) RNA molecules. molecules.

► Cleavage clearly involves virus- specified proteases, Cleavage clearly involves virus- specified proteases, and the polyprotein itself is enzymatically active in and the polyprotein itself is enzymatically active in Trans.Trans.

► Two or more subgenomic mRNA species are made in Two or more subgenomic mRNA species are made in cells infected with corona, calici or HE virusescells infected with corona, calici or HE viruses..

Flow of events during the Flow of events during the replication of Togaviruses. replication of Togaviruses.

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

► The consequences are two foldThe consequences are two fold::

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

► Second, because all (+) strand genomes are Second, because all (+) strand genomes are monopartite, the initial products of translation monopartite, the initial products of translation of both genomic RNA and mRNA species are of both genomic RNA and mRNA species are necessarily a single protein. necessarily a single protein.

3- Retroviruses3- Retroviruses► First step in replication is synthesis of a DNA First step in replication is synthesis of a DNA

strand complementary to the RNA genome, strand complementary to the RNA genome, followed by digestion of RNA by a nuclease followed by digestion of RNA by a nuclease (ribonuclease H in the virion), and finally (ribonuclease H in the virion), and finally synthesis of a complementary DNA strand.synthesis of a complementary DNA strand.

► The linear ds DNA translocated into the nucleus The linear ds DNA translocated into the nucleus integrates into the host genome (Provirus). integrates into the host genome (Provirus).

► The products of transcription are genome-The products of transcription are genome-length RNA molecules (efficiently packaged into length RNA molecules (efficiently packaged into virions), and shorter, spliced mRNAs that are virions), and shorter, spliced mRNAs that are translated to yield polyproteins that are translated to yield polyproteins that are processed by cleavage to individual viral processed by cleavage to individual viral proteinsproteins..

Flow of events during the Flow of events during the replication of retroviruses.replication of retroviruses.

4- Non segmented Negative (-) strand RNA 4- Non segmented Negative (-) strand RNA virusesviruses

► They have their transcriptases packaged in the They have their transcriptases packaged in the virion. virion.

► The transcription of the viral genome is the first The transcription of the viral genome is the first event after entry into cells (multiple functional event after entry into cells (multiple functional mRNAs are produced).mRNAs are produced).

► Replication begins under the direction of newly Replication begins under the direction of newly

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

5- Segmented Negative strand RNA viruses5- Segmented Negative strand RNA viruses

► The first step involves the synthesis of mRNAs The first step involves the synthesis of mRNAs from each segment of the genomic RNA.from each segment of the genomic RNA.

► The mRNAs of influenza virus have heterogeneous The mRNAs of influenza virus have heterogeneous nonviral 5nonviral 5’’ end sequences (8 end sequences (8 –– 18 nucleotides ) 18 nucleotides ) that are stolenthat are stolen”” from the host cell mRNA from the host cell mRNA molecules by viral proteinsmolecules by viral proteins..

► The newly synthesized viral proteins replicate the The newly synthesized viral proteins replicate the genomic RNA segments to yield precise (+) strand genomic RNA segments to yield precise (+) strand copies of the virion RNAscopies of the virion RNAs

► A unique characteristic of them is reassortment of A unique characteristic of them is reassortment of

their genes in cells infected by more than one their genes in cells infected by more than one virion of the same group introducing new virion of the same group introducing new genotypesgenotypes..

Flow of events during the replication Flow of events during the replication of Orthomyxoviruses and of Orthomyxoviruses and

ParamyxovirusesParamyxoviruses..

► The genes of (-) strand viruses serves as The genes of (-) strand viruses serves as template for transcription only.template for transcription only.

► The consequences are three- foldThe consequences are three- fold:-:-

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

► Second, naked RNA extracted from virions is not Second, naked RNA extracted from virions is not infectious .infectious .

► Third, mRNAs produced are gene unit length, they Third, mRNAs produced are gene unit length, they specify a single polypeptide.specify a single polypeptide.

► Consequently, the (+) transcript which functions as Consequently, the (+) transcript which functions as mRNA is different form the (+) strand RNA which serves mRNA is different form the (+) strand RNA which serves as the template for progeny virus even though both are as the template for progeny virus even though both are synthesized on the genomic RNAsynthesized on the genomic RNA..

6-6- Ambisenes RNA VirusesAmbisenes RNA Viruses (Arenaviruses and Bunyaviruses)(Arenaviruses and Bunyaviruses)

► The expression of this information takes place The expression of this information takes place in two stages. in two stages.

► The genomic RNA is transcribed to yield (+) strand The genomic RNA is transcribed to yield (+) strand subgenomic size mRNA. subgenomic size mRNA.

► The appropriate full size complementary RNA is then The appropriate full size complementary RNA is then transcribed to yield subgenomic size mRNA. transcribed to yield subgenomic size mRNA.

► Because the replicative cycles begin with the Because the replicative cycles begin with the transcription of genomic RNA, the ambisense transcription of genomic RNA, the ambisense viruses must carry their own polymerase into viruses must carry their own polymerase into the infected cellthe infected cell..

7- 7- Double Stranded RNA viursesDouble Stranded RNA viurses

► The multipartite reovirus genome is transcribed within The multipartite reovirus genome is transcribed within the partially opened capsid by a polymerase packaged the partially opened capsid by a polymerase packaged into the virioninto the virion

► The 10 mRNA (+) strand species are extruded from the The 10 mRNA (+) strand species are extruded from the exposed vertices of the capsidexposed vertices of the capsid..

► The mRNA molecules have two functions: The mRNA molecules have two functions:

► first, they are translated as monocistronic messages to first, they are translated as monocistronic messages to yield the viral proteins. yield the viral proteins.

► Second, one RNA of each of the 10 species assemble Second, one RNA of each of the 10 species assemble within a precursor of particle in which it servers as a within a precursor of particle in which it servers as a template for synthesis of the complementary strand, template for synthesis of the complementary strand, yielding ds genome segmentsyielding ds genome segments..

Flow of events during the replication of Flow of events during the replication of

ReovirusesReoviruses

►II- DNA VirusesII- DNA Viruses 1- Double – Stranded DNA Viruses that Replicate 1- Double – Stranded DNA Viruses that Replicate

in the Nucleusin the Nucleus

► Significant differences exist in the replication strategies Significant differences exist in the replication strategies of Nuclear virusesof Nuclear viruses..

► Papovaviruses encode a single protein that binds in Papovaviruses encode a single protein that binds in close proximity to the origin of viral DNA synthesis, close proximity to the origin of viral DNA synthesis, stimulates the cellular polymerase complex to replicate stimulates the cellular polymerase complex to replicate the viral DNA, and acts as a helicase. the viral DNA, and acts as a helicase.

► Adenoviruses encode a DNA polymerase but depend on Adenoviruses encode a DNA polymerase but depend on the host cells for all other functions involved in the the host cells for all other functions involved in the synthesis of their DNA. synthesis of their DNA.

► At The other extreme are the herpesviruses; HSV At The other extreme are the herpesviruses; HSV encodes numerous proteins involved in the pathway of encodes numerous proteins involved in the pathway of the synthesis of DNAthe synthesis of DNA . .

Flow of events during the replication Flow of events during the replication of herpesviruses (herpes simplex of herpesviruses (herpes simplex

viruses).viruses).

2- Double stranded DNA Viruses that replicate 2- Double stranded DNA Viruses that replicate in the cytoplasmin the cytoplasm

► Transcriptional events and most of the other events Transcriptional events and most of the other events in the reproductive cycle seem to take place in the in the reproductive cycle seem to take place in the cytoplasm.cytoplasm.

► Poxviurses have evolved all of the factors necessary Poxviurses have evolved all of the factors necessary for transcription and replication of their genome.for transcription and replication of their genome.

► Because host transcriptional factors are not Because host transcriptional factors are not involved, the cis - acting sites for the synthesis and involved, the cis - acting sites for the synthesis and processing of the mRNA have diverged from those processing of the mRNA have diverged from those of the host. of the host.

► The initial transcription occurs in the core of the The initial transcription occurs in the core of the virion, the protein products of these transcripts virion, the protein products of these transcripts function to release the viral genome from the core. function to release the viral genome from the core.

3- Single- stranded DNA viruses3- Single- stranded DNA viruses (Parvoviruses)(Parvoviruses)

► Multiplication requires the synthesis of a DNA Multiplication requires the synthesis of a DNA strand complementary to the ss gnomic DNA in strand complementary to the ss gnomic DNA in the nucleus and transcription of the genomethe nucleus and transcription of the genome..

► The B19 virus replicates in mitotically active cells The B19 virus replicates in mitotically active cells and prefers cells of the erythroid lineage.and prefers cells of the erythroid lineage.

► Factors available only during the S phase of the Factors available only during the S phase of the

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

► A ds DNA version of the virion genome is A ds DNA version of the virion genome is

required for transcription and replicationrequired for transcription and replication..

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

► The palindromic sequence (about 115 bases at The palindromic sequence (about 115 bases at both ends) can fold back on it self and forms ds both ends) can fold back on it self and forms ds sequences stabilized by hydrogen bonding in sequences stabilized by hydrogen bonding in the form of hairpin Y or T shapethe form of hairpin Y or T shape..

► The ds DNA replicative intermediate is The ds DNA replicative intermediate is transcribed by cellular RNA polymerases and transcribed by cellular RNA polymerases and replicated by DNA polymerasereplicated by DNA polymerase..

► In the absence of a helper virus, the genomes In the absence of a helper virus, the genomes of dependent parvovirus appear to integrate of dependent parvovirus appear to integrate into a specific locus on a human chromosome into a specific locus on a human chromosome

Flow of events during the replication Flow of events during the replication of Parvovirusesof Parvoviruses

4- Hepadnaviruses4- Hepadnaviruses

► Hepadnaviruses have a circular partially ds Hepadnaviruses have a circular partially ds DNA genome. They replicate in the nucleus. DNA genome. They replicate in the nucleus.

► The gap in the DNA of the virus is repaired The gap in the DNA of the virus is repaired first by a DNA polymerase packaged into first by a DNA polymerase packaged into virion. virion.

► the genome is then transcribed into two the genome is then transcribed into two classes of RNA molecules; RNAs specifying classes of RNA molecules; RNAs specifying proteins and a full length RNA that serves proteins and a full length RNA that serves as a template for the synthesis of genomic as a template for the synthesis of genomic DNA by a virally encoded reverse DNA by a virally encoded reverse transcriptasetranscriptase..

Flow of events during the replication Flow of events during the replication

of Hepadnaviruses (hepatitis B virus).of Hepadnaviruses (hepatitis B virus).

Assembly, Maturation, and Egress of Assembly, Maturation, and Egress of viruses from infected cellsviruses from infected cells

►Assembly of DNA viruses, except Assembly of DNA viruses, except poxviruses, occurs in the nucleus and poxviruses, occurs in the nucleus and requires transport of the virion proteins requires transport of the virion proteins into the nucleus. into the nucleus.

►Assembly of pox and RNA viruses takes Assembly of pox and RNA viruses takes place in the cytoplasm.place in the cytoplasm.

► The assembly process begins when the The assembly process begins when the

concentration of structural proteins in the concentration of structural proteins in the cell is sufficient to thermodynamically cell is sufficient to thermodynamically drive the process, much like a drive the process, much like a crystallization reactioncrystallization reaction..

► Structural proteins of simple icosahedral Structural proteins of simple icosahedral viruses can aggregate spontaneously to viruses can aggregate spontaneously to from structural units, which in turn from structural units, which in turn assemble into empty capsids (procasids). assemble into empty capsids (procasids).

► Somehow, the viral nucleic acid now Somehow, the viral nucleic acid now enters this structure via a mechanism that enters this structure via a mechanism that seems to involve a nucleotide sequence seems to involve a nucleotide sequence known as the “packing sequence”. known as the “packing sequence”.

►Helical viruses assemble by adding blocks Helical viruses assemble by adding blocks during coiling of the viral nucleic acid. during coiling of the viral nucleic acid.

►Maturation and release are determined in part Maturation and release are determined in part by site of replication and the presence of an by site of replication and the presence of an envelope. envelope.

► Acquisition of an envelope occurs after Acquisition of an envelope occurs after association of the nucleocapsid with regions of association of the nucleocapsid with regions of host cell membrane modified by matrix protein host cell membrane modified by matrix protein and glycoproteins. and glycoproteins.

►Matrix proteins line and promote the adhesion Matrix proteins line and promote the adhesion of nuclecocapsids with the modified membrane. of nuclecocapsids with the modified membrane.

► As more interactions occur, the membrane As more interactions occur, the membrane surrounds the nucleocapsid and the virus buds surrounds the nucleocapsid and the virus buds from the membranefrom the membrane . .

strategies for maturationstrategies for maturation► Three fundamental strategies for maturation Three fundamental strategies for maturation

have been describedhave been described:-:-

I- Intracellular assembly and MaturationI- Intracellular assembly and Maturation - Nonenveloped viruses cause disintegration of the - Nonenveloped viruses cause disintegration of the infected cell for their egress.infected cell for their egress.

II- Strategy of enveloped virusesII- Strategy of enveloped viruses

-The last step in assembly of (-) strand RNA viruses -The last step in assembly of (-) strand RNA viruses is linked with their egress from infected cells by is linked with their egress from infected cells by budding from the cytoplasmic or other membranes.budding from the cytoplasmic or other membranes.

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

► They range from highly cytolytic (toga, They range from highly cytolytic (toga, paramyxo) to viruses which are frequently paramyxo) to viruses which are frequently noncytolytic noncytolytic

(retroviruses) .(retroviruses) .

► By virtue of the viral glycoprotein insertion into By virtue of the viral glycoprotein insertion into the cell surface, however, these viruses import the cell surface, however, these viruses import upon the cell a new antigenic specificity and upon the cell a new antigenic specificity and the infected cell can and does become a target the infected cell can and does become a target for the immune mechanisms of the host.for the immune mechanisms of the host.

III- Strategy for HerpesvirusesIII- Strategy for Herpesviruses

- - They assemble their nucleocapsid in the They assemble their nucleocapsid in the nucleusnucleus.. - - Envelopment and maturation occur at Envelopment and maturation occur at the inner lamella of the nuclear the inner lamella of the nuclear

membranemembrane

- - Herpesvirurses are cytolytic and Herpesvirurses are cytolytic and invariably destroy the cell in which they invariably destroy the cell in which they

multiply. multiply.

- - They also import new antigens on the They also import new antigens on the infected cell. infected cell.

Glycosylation and BuddingGlycosylation and Budding

► In the glycosylation of their proteins, viruses In the glycosylation of their proteins, viruses use existing pathways.use existing pathways.

► This involves a “signal sequence“ of 15-30 This involves a “signal sequence“ of 15-30 hydrophobic amino acids that facilitate binding hydrophobic amino acids that facilitate binding to a receptor on the cytoplasmic side of the to a receptor on the cytoplasmic side of the RER. RER.

► It then passes through the lipid bilayer to the It then passes through the lipid bilayer to the luminal side where the signal sequences is luminal side where the signal sequences is removed by a signal peptidase allowing the removed by a signal peptidase allowing the addition of oligosaccharides. addition of oligosaccharides.

►Glucose is then removed by glucosidase Glucose is then removed by glucosidase (trimming).(trimming).

► The viral glycoprotein is then transported to the The viral glycoprotein is then transported to the

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

► Another coated vesicle now transports the Another coated vesicle now transports the acylated glycoprotein to the plasma membrane acylated glycoprotein to the plasma membrane or cytoplasmic structures, probably with the or cytoplasmic structures, probably with the help of a leading sequence that finds the help of a leading sequence that finds the destination (postal address or zip codedestination (postal address or zip code((..

► Envelope glycoproteins are then cleaved into 2 Envelope glycoproteins are then cleaved into 2 poly- peptide chains that remain covalently poly- peptide chains that remain covalently bound by S-S bonds. bound by S-S bonds.

► Then the hydrophilicThen the hydrophilic N-terminus of the N-terminus of the glycoprotein finds itself projecting from the glycoprotein finds itself projecting from the external surface of the membrane while the external surface of the membrane while the hydrophobic domain near the c-terminus hydrophobic domain near the c-terminus remains anchored in the lipid bilayerremains anchored in the lipid bilayer..

► Budding is a form of exocytosis (reversed Budding is a form of exocytosis (reversed endocytosis) and viruses remain cell- endocytosis) and viruses remain cell- associated for few hours and large numbers of associated for few hours and large numbers of viruses are released in consecutive wavesviruses are released in consecutive waves..

Variability in viral Genomes and viral Variability in viral Genomes and viral MultiplicationMultiplication

►On passage, viruses tend to yield defective On passage, viruses tend to yield defective mutants.mutants.

► It is convenient to classify defective viruses into It is convenient to classify defective viruses into two groups. two groups.

Viruses in the first group lack one or more Viruses in the first group lack one or more essential genes and therefore are incapable of essential genes and therefore are incapable of independent replication without a helper virus.independent replication without a helper virus.

- They can transform infected cells or - They can transform infected cells or

transactivate oncogenic viruses in causing the transactivate oncogenic viruses in causing the cell to become malignantcell to become malignant..

The second group comprises viruses which contain The second group comprises viruses which contain mutations and deletions and therefore cannot mutations and deletions and therefore cannot replicate in an efficient fashion. replicate in an efficient fashion.

- Chronic debilitating infections of the CNS might in - Chronic debilitating infections of the CNS might in some fashion be related to viruses that are sluggish some fashion be related to viruses that are sluggish in their replication, in their ability to destroy the in their replication, in their ability to destroy the infected cells, or in their ability to alter the infected infected cells, or in their ability to alter the infected cell sufficiently to make it a target for the immune cell sufficiently to make it a target for the immune system of the hostsystem of the host....

- Genetically, engineered viruses lacking one or - Genetically, engineered viruses lacking one or several genes and which might be classified as several genes and which might be classified as defective may ultimately be viruses greatest gift to defective may ultimately be viruses greatest gift to mankind; the means for the introduction of genes to mankind; the means for the introduction of genes to complement genetic deficits or to selectively destroy complement genetic deficits or to selectively destroy cancer cellscancer cells..