Replication of Positive-Sense RNA Viruses
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Replication of Positive-Replication of Positive-Sense RNA VirusesSense RNA Viruses
Replication of Positive-Replication of Positive-Sense RNA VirusesSense RNA Viruses
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Virus Replication
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RNA Virus Replication
• Replicative Intermediate 1– Genome template– Makes Antigenome
(opposite sense)• Replicative
Intermediate 2– Antigenome
template– Makes new
Genome for progeny virus
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Family: Picornaviridae• “small”• (+)RNA virus• Nonenveloped• Icosahedral capsid, 30 nm
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Genus: Enterovirus• “intestine”• Large group of related viruses:
– Poliovirus (poliomyelitis -“gray”, “marrow”)– Echovirus (enteric cytopathic human
orphan)– Coxsackievirus– Enterovirus
• Infect URT, GI, CNS, heart, muscle, skin• pH stable
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Genus: Rhinovirus• “nose”• Human rhinovirus (>100
serotypes)• Common cold viruses• Acid pH labile
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Genus: Aphthovirus• “ulcer”• Foot-and-mouth disease virus
(FMDV) of livestock• Infect cattle, sheep, pigs, goats
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Genus: Cardiovirus• “heart”• Encephalomyocarditis virus (EMC)
of mice• Infect brain, muscle, heart
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Genus: Heparnavirus• “liver”• Hepatitis A virus• “infectious” hepatitis• Transmission via contaminated
food or water
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Poliovirus: (+)RNA Genome
• 7.7 kb• Single open
translational reading frame (ORF)
• 5’ end has small viral protein (VPg)
• 3’ polyA tail• Serves directly as
mRNA (“infectious”)
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Poliovirus: RNA Genome Expression
• Attachment and entry by receptor-mediated endocytosis
• Uncoat and release of RNA into cytoplasm
• RNA attaches to cell ribosome - recognition by Internal ribosome entry site (IRES) sequence on 5’ end of genome RNA
• Viral mRNA translates for one large polyprotein
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Poliovirus Polyprotein• Single polyprotein cleaved by viral
protease (2A, 3C) into 3 precursor proteins (P-1, P-2, P-3)
• Precursor proteins cleaved further by viral protease
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Poliovirus Proteins
• P-1: capsid proteins (VP4, VP2, VP3, VP1)• P-2: protease (2A), regulatory proteins (2B, 2C)
• P-3: regulatory protein (3A), VPg, protease (3C), RNA polymerase (3D)
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Poliovirus: Transcription of Complimentary RNA (RI-1)
• Antigenome copied from released (+)RNA genome
• Transcription complex:– (+)RNA genome– VPg serves as
primer (attaches to 3’ end of viral RNA)
– Host proteins required
– Viral RNA Replicase (viral protein 3D)
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Poliovirus: Replication of Genome RNA (RI-2)
• (+)RNA genome copied from Antigenome template
• Transcription complex: antigenome, VPg, host proteins, viral RNA replicase
• New (+)RNA serves as:– mRNA (more viral proteins)– genome RNA (for new
virus)
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Poliovirus RNA
Synthesis
• Each cell makes ~ 4 x 105 RNA molecules
• (-)RNA ~ 10%• (+)RNA ~
90%
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Poliovirus: Assembly
• Protomer (VP0, VP1, VP3)
• Pentamer (five protomers)
• Procapsid (twelve pentamers)
• Provirion (sixty protomers, “head-full” insertion of viral RNA genome)
• Virion (cleavage of VP0 into VP2, VP4)
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Poliovirus: Cell Cytopathic Effects
(CPE)• Host cell translation stopped shortly (30
min.) after infection• Viral protease activates host cell
protease to cleave a 220 kd cell ribosome protein of eIF-4F
• No cap-binding complex of mRNA• No attachment of host mRNA to
ribosome• No cell protein synthesis, leads to cell
death
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Poliovirus: Release By Cell Lysis
• Occurs within 5-10 hours in cell culture
• Each cell releases ~ 25 – 100,000 virus particles
• Not all virus particles are infectious
• Virus spreads to other cells
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“The fight against infantile paralysis is a fight to the finish, and the terms
are unconditional surrender.”• Franklin D. Roosvelt, 32nd U.S.
President and polio survivor, 1944
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Family: Flaviviridae• “yellow”• (+)RNA, 10 kb
– Single ORF– 5’ cap, 3’ polyA
tail– Translates for
single polyprotein, cleaved into structual (capsid, M, E) and non-structual (NS) proteins
• Enveloped• Icosahedral, 40-
50 nm• Replicate in
arthropods, animals, humans
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Genus: Flavivirus
• Former “Group B Arboviruses” (animal to human transmission by arthropod bite)
• Yellow fever virus (monkeys)
• West Nile virus (birds)• St. Louis encephalitis
virus (birds)• Hepatitis C virus
(transmission by blood and body secretions)
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Family: Togaviridae• “coat”• (+)RNA virus• Enveloped• Icosahedral capsid, 60-70 nm• Replicate in arthropods, animals,
humans
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Genus: Alphavirus
• Former “Group A Arboviruses”• Sindbis virus (bird, monkey, rare
mild disease in human)• Equine encephalitis virus (horse,
bird, rodent)
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Genus: Rubivirus• “red”• Rubella virus• “3 day measles”• Transmission by direct contact, aerosols• Mild skin rash disease, but infected
moms may transmit to fetus (congenital defects, death)
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Sindbis Virus: (+)RNA Genome
• Genome (11 kb), two ORF, 5’ cap, 3’ polyA tail
• Genome RNA (11 kb) translates ORF-1
• Synthesis of a subgenomic mRNA (4 kb) to translate ORF-2
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Sindbis Virus: “Early” Gene Expression
• Attachment, endocytosis, uncoat and release of (+)RNA into cytoplasm
• RNA binds to ribosome and translates ORF-1 for a polyprotein
• Polyprotein cleaved into four nonstructural proteins
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Sindbis Virus: Transcription
Complementry RNA (RI-1)
• Antigenome copied from released viral Genome RNA
• By “early” viral replication enzymes
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Sindbis Virus: Replication
Genome RNA(RI-2)
• Genome (+)RNA (49s) copied from Antigenome
• By viral replication enzymes
• New RNA serves as:– mRNA– Genome RNA
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Sindbis Virus: “Late” Gene Expression
• Viral RNA replicase binds to Interior replicase start site of antigenome
• Synthesis of a subgenomic mRNA (26s), 5’ Cap, 3’ PolyA
• Translation of ORF-2 for structural (capsid, envelope) proteins
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Sindbis Virus RNA• Temporal regulation of viral RNA
synthesis• 1–3 hours: both (+)RNA and (-)RNA• >3 hours: mainly (+)RNA• 10x more subgenomic mRNA (26s)
than genomic mRNA (49s)
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Sindbis Virus: Assembly and Release By Budding
• Envelope proteins glycosylated in ER/Golgi, transported and inserted into plasma membrane
• Capsid forms in cytoplasm
• Bud through the virus modified plasma membrane, picks up envelope, and released from cell
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Family: Coronaviridae
• Corona - “crown”• Envelope, large petal
spikes, 80-120 nm• Flexible helical
nucelocapsid, 10-20 nm• (+)RNA, 30 kb, 5’ cap,
3’ polyA, five ORFs• Synthesis of
subgenomic mRNAs
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Genus: Coronavirus• Human coronavirus (common cold,
LRTI, gastroenteritis)• SARS-CoV (severe acute
respiratory syndrome, fatal pneumonia)
• Also infections of mammals and birds
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Reading & Questions• Chapter 14: Replication of
Positive-Sense RNA Viruses
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QUESTIONS???
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Class Discussion – Lecture 5
• 1. Why is the RNA genome of poliovirus by itself “infectious” when transfected into a host cell?
• 2. How does (+)RNA viruses replicate their genome via two replicative intermediates? Why is this necessary?
• 3. Is the replication strategy of Sindbis virus similar/different from poliovirus?
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MICR 401 FIRST EXAM• Tuesday, Oct. 16, 2012• History through Positive-sense RNA
virus: Coronavirus• Lecture, Discussion Questions, Reading,
Chapter Questions• Test Format:
– Objective questions – Multiple Choice, True/False Statement, Identification (term, figure, diagram)
– Short essay questions (similar to Class Discussion Questions and Basic Virology Chapter Questions)