viral replication
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Viral Replication. Scott M. Hammer, M.D. Viral Replication: Basic Concepts. Viruses are obligate intracellular parasites Viruses carry their genome (RNA or DNA) and sometimes functional proteins required for early steps in replication cycle - PowerPoint PPT PresentationTRANSCRIPT
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Viral Replication
Scott M. Hammer, M.D.
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Viral Replication: Basic Concepts
• Viruses are obligate intracellular parasites
• Viruses carry their genome (RNA or DNA) and sometimes functional proteins required for early steps in replication cycle
• Viruses depend on host cell machinery to complete replication cycle and must commandeer that machinery to successfully replicate
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Viral Replication: Basic Concepts
• Replication cycle produces- Functional RNA’s and proteins- Genomic RNA or DNA and structural proteins
• 100’s-1,000’s new particles produced by each cycle- Referred to as burst size- Many are defective- End of ‘eclipse’ phase
• Replication may be cytolytic or non-cytolytic
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Steps in Viral Replication: Attachment(First Step)
• Surface protein on virus attaches to specific receptor(s) on cell surface- May be specialized proteins with limited tissue
distribution or more widely distributed- Virus specific receptor is necessary but not sufficient
for viruses to infect cells and complete replicative cycle
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Selected Virus Receptors
AdenovirusCoxsackievirus
EchovirusEpstein-Barr Virus
HIV-1Measles virus
ParvovirusPoliovirus
Rhinovirus
CARCAR, CD55Integrin VLA-2, CD55CD21CD4, CCR5, CXCR4CD46Erythrocyte P AgPVRICAM-1
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Steps in Viral Replication: Penetration(Second Step)
• Enveloped viruses penetrate cells through fusion of viral envelope with host cell membrane- May or may not involve receptor mediated endocytosis
• Non enveloped viruses penetrate by- Receptor mediated endocytosis - Translocation of the virion across the host cell
membrane
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Influenza Virus Replication Cycle
From Fields Virology
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Steps in Viral Replication: Uncoating(Third Step)
• Makes viral nucleic acid available for transcription to permit multiplication to proceed
• Mechanism variably understood depending upon the virus
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Uncoating of Influenza Virus
From Fields Virology
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Steps in Viral Replication: Basic Strategies of Transcription and Translation
(Fourth and Fifth Steps)
• (+) RNA Proteins
• (-) RNA (+) RNA Proteins
• RNA DNA RNA Proteins
• DNA RNA Proteins
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Steps in Viral Replication: Assembly and Release
(Sixth and Seventh Steps)
• Process involves bringing together newly formed genomic nucleic acid and structural proteins to form the nucleocapsid of the virus
• Nonenveloped viruses exhibit full maturation in the cytoplasm or nucleus with disintegration of cell
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Steps in Viral Replication: Assembly and Release
(Sixth and Seventh Steps)
• Many enveloped viruses exhibit full maturation as the virion exits the cell- Viral proteins are inserted into the host cell membrane- Nucleocapsids bind to these regions and bud into the
extracellular space- Further cleavage and maturation of proteins may occur
after viral extrusion- Cytolytic activity of these viruses varies
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Influenza Virus
From Fields Virology
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Retroviruses
From Fields Virology
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Steps in Viral Replication: Assembly and Release
(Sixth and Seventh Steps)
• Herpesviruses (enveloped) assemble nucleocapsids in the nuclei of infected cells and mature at the inner lamella of the nuclear membrane- Virions accumulate in this space, in the ER and in
vesicles- Virion release is associated with cytolysis
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Herpes Simplex Virus
From Fields Virology
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Schematic of Replication Cycle of (+) RNA Single Strand Viruses Coding for One Sized RNA
Genomic RNA binds toribosomes and is translatedinto polyprotein
Polyprotein is cleaved
Genomic RNA’s serve as templates for synthesis ofcomplementary full length(-) RNA’s by viral polymerase
(-) strand RNA serves astemplate for (+) strand RNA’s;these serve to produce morepolyprotein, more (-) strand RNA’s or become part of newvirions
From Fields Virology
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Schematic of Replication Cycle of (+) RNA Single Strand Viruses Coding for
Genomic and Subgenomic RNA’s
Genomic RNA binds toribosomes but only a portionof 5’ end is translated into non-structural proteins
(-) strand RNA is synthesized.Different classes of (+) RNA’sare produced. One is trans-lated into a polyprotein whichis cleaved to form structuralproteins. Another is full lengthand serves as genomic RNA for new virions
From Fields Virology
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Schematic of Nonsegmented (-) RNA Strand Virus Replication Cycle
Transcription of (-) strand occursafter entry and mediated by virionpackaged transcriptase
(+) strand RNA’s produced; proteinssynthesized
Full length (-) strand RNA’sproduced and packaged into newvirions
Transcription and translation takeplace entirely in cytoplasm
From Fields Virology
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Schematic of Segmented (-) RNA Strand Virus Replication Cycle
mRNA’s are synthesized fromeach segment
Viral proteins are synthesized
(+) strand RNA’s are synthesizedand serve as templates for(-) strand genomic RNA’s
From Fields Virology
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Schematic of Double Strand RNA Virus Replication Cycle
Genome transcribed by virionpackaged polymerase
mRNA’s are translated to proteinsor transcribed to complementaryRNA strands to yield DS RNAgenomes for new virions
From Fields Virology
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Schematic of Herpesvirus Replication Cycle(DS DNA Virus Which Replicates in Nucleus)
Sequential, orderedrounds of mRNA andprotein productionregulate replication
Structural proteins produced during lastcycle of replication
From Fields Virology
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Schematic of Partially Double Stranded DNA Virus Replication Cycle
(e.g., hepatitis B virus)Genome of hepatitis B is circularand partially double stranded; itis replicated in nucleus
Genome converted to closed circular molecule by DNApolymerase which is virion packaged
Two classes of RNA species areproduced: one that codes for viral proteins and one thatproduces genomic DNA by avirally encoded RT
From Fields Virology
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Retrovirus Virion
From Fields Virology
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Genomic Structure of Primate Lentiviruses
From Fields Virology
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Retrovirus Replication Cycle
From Fields Virology
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HIV Entry
FusionComplete
CD4Attachment
HR1-HR2interaction
CXCR4CCR5
HIV
HIVgp120
Anchorage
CD4
Co-receptorinteraction
Cell
HIV
HIV
HIV
gp41
gp41
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HIV Tat and Rev Function
From Fields Virology
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Primary HIV Infection:Pathogenetic Steps
• Virus – dendritic cell interaction- Infection is typically with R5 (M-tropic) strains- Importance of DC-SIGN
• Delivery of virus to lymph nodes• Active replication in lymphoid tissue• High levels of viremia and dissemination• Downregulation of virus replication by immune
response• Viral set point reached after approximately 6
months
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PHI: Early Seeding of Lymphoid Tissue
Schacker T et al: J Infect Dis 2000;181:354-357
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Primary HIV Infection:Clinical Characteristics
• 50-90% of infections are symptomatic• Symptoms generally occur 5-30 days after
exposure• Symptoms and signs
- Fever, fatigue, myalgias, arthralgias, headache, nausea, vomiting, diarrhea
- Adenopathy, pharyngitis, rash, weight loss, mucocutaneous ulcerations, aseptic meningitis, occas. oral/vaginal candidiasis
- Leukopenia, thrombocytopenia, elevated liver enzymes• Median duration of symptoms: 14 days
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The Variable Course of HIV-1 InfectionTypical Progressor Rapid Progressor
Vira
l Rep
licat
ion
CD
4 Level
months years
Primary HIVInfection Clinical Latency AIDS
A Vira
l Rep
licat
ion
CD
4 Level
months years
Primary HIVInfection AIDS
BVi
ral R
eplic
atio
n CD
4 Level
months years
Primary HIVInfection Clinical Latency
C
Nonprogressor
?Reprinted with permission from Haynes. In: DeVita et al, eds. AIDS: Etiology, Treatment and Prevention.4th ed. Lippincott-Raven Publishers; 1997:89-99.
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Primary HIV Infection: Determinants of Outcome
• Severity of symptoms• Viral strain
- SI (X4) vs. NSI (R5) viruses• Immune response
- CTL response- Non-CTL CD8 responses- Vβ repertoire pattern- ADCC- Humoral responses?
• Viral set point at 6-24 months post-infection• Other host factors
- Chemokine receptor and HLA genotype• Gender and differences in viral diversity?• Antiviral therapy
- Near vs. long-term benefit?
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Natural History of Untreated HIV-1 Infection
Time in YearsInfection
CD4Cells
1000
800
600
400
200
0
Early Opportunistic Infections
Late Opportunistic Infections+
1 2 3 4 5 6 7 8 9 10 11 12 13 14
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RNARNA
ReverseReversetranscriptasetranscriptase
ProteaseProtease
DNADNA
NucleusNucleus
Protease inhibitorsProtease inhibitorsReverse transcriptase inhibitorsReverse transcriptase inhibitors
Antiviral Agents for HIVAntiviral Agents for HIV
EntryEntryInhibitorsInhibitors
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---- - -- -- -- - -- --- - - -- -- -- ----- -- - ----- -- -- --- -- -- ----- - ----- - -- -- --- -- ---- - -- --- - ------- - -- -- --- -- --- - - -- - --- - ---- -- - -- - -- - --- -- -- --- ---- ---
gp41
gp120
---- - -- -- -- - -- --- - - -- -- -- ----- -- - ----- -- -- --- -- -- ----- - ----- - -- -- --- -- ---- - -- --- - ------- - -- -- --- -- --- - - -- - --- - ---- -- - -- - -- - --- -- -- --- ---- ---
---- - -- -- -- - -- --- - - -- -- -- ----- -- - ----- -- -- --- -- -- ----- - ----- - -- -- --- -- ---- - -- --- - ------- - -- -- --- -- --- - - -- - --- - ---- -- - -- - -- - --- -- -- --- ---- ---
---- - -- -- -- - -- --- - - -- -- -- ----- -- - ----- -- -- --- -- -- ----- - ----- - -- -- --- -- ---- - -- --- - ------- - -- -- --- -- --- - - -- - --- - ---- -- - -- - -- - --- -- -- --- ---- ---
ReceptorBinding
Native Form
Fusion peptide
HR1
HR2
FusionIntermediate
T20
---- - -- -- -- - -- --- - - -- -- -- ----- -- - ----- -- -- --- -- -- ----- - ----- - -- -- --- -- ---- - -- --- - ------- - -- -- --- -- --- - - -- - --- - ---- -- - -- - -- - --- -- -- --- ---- ---
“Ensnared” Transition State Intermediate
Conformation
FusionBlockade
Membrane Fusion
Core Structure
T1249
Mechanism of T20/T1249Mediated Fusion InhibitionModified from Weissenhorn et al., Nature 387, 426-430 (1997) and Furuta et al., Nature structural biology 5, 276-279 (1998).
Conformation
X
---- - -- -- -- - -- --- - - -- -- -- ----- -- - ----- -- -- --- -- -- ----- - ----- - -- -- --- -- ---- - -- --- - ------- - -- -- --- -- --- - - -- - --- - ---- -- - -- - -- - --- -- -- --- ---- ---
Cell Membrane
Virus Membrane