Replication of Positive-Sense RNA Viruses

1. Replication of Positive-Sense RNA Viruses

2. Virus Replication

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

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

5. 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

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

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

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

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

10. 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”)

11. 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

12. 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

13. 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)

14. 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)

15. 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)

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

17. 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)

18. 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

19. 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

20. “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

21. 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

22. 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)

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

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

25. 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)

26. 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

27. 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

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

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

30. 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

31. 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)

32. 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

33. 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

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

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

36. QUESTIONS???

37. 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?

38. 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)