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 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, 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, 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, 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)