Replication of Positive-Sense RNA Viruses

Virus Replication

RNA Virus Replication Replicative Intermediate 1 Genome template Makes Antigenome (opposite sense) Replicative Intermediate 2 Antigenome template Makes new Genome for progeny virus

Family: Picornaviridae “small” (+)RNA virus Nonenveloped Icosahedral capsid, 30 nm

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

Genus: Rhinovirus “nose” Human rhinovirus (>100 serotypes) Common cold viruses Acid pH labile

Genus: Aphthovirus “ulcer” Foot-and-mouth disease virus (FMDV) of livestock Infect cattle, sheep, pigs, goats

Genus: Cardiovirus “heart” Encephalomyocarditis virus (EMC) of mice Infect brain, muscle, heart

Genus: Heparnavirus “liver” Hepatitis A virus “infectious” hepatitis Transmission via contaminated food or water

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”)

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

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

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)

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)

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)

Poliovirus RNA Synthesis Each cell makes ~ 4 x 105 RNA molecules (-)RNA ~ 10% (+)RNA ~ 90%

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)

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

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

“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

Family: Flaviviridae “yellow” (+)RNA, 10 kb Enveloped 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

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)

Family: Togaviridae “coat” (+)RNA virus Enveloped Icosahedral capsid, 60-70 nm Replicate in arthropods, animals, humans

Genus: Alphavirus Former “Group A Arboviruses” Sindbis virus (bird, monkey, rare mild disease in human) Equine encephalitis virus (horse, bird, rodent)

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)

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

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

Sindbis Virus: Transcription Complementry RNA (RI-1) Antigenome copied from released viral Genome RNA By “early” viral replication enzymes

Sindbis Virus: Replication Genome RNA (RI-2) Genome (+)RNA (49s) copied from Antigenome By viral replication enzymes New RNA serves as: mRNA Genome RNA

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

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)

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

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

Genus: Coronavirus Human coronavirus (common cold, LRTI, gastroenteritis) SARS-CoV (severe acute respiratory syndrome, fatal pneumonia) Also infections of mammals and birds

Reading & Questions Chapter 14: Replication of Positive-Sense RNA Viruses

QUESTIONS???

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?

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)