Replication of Double-Stranded RNA Virus; Subviral Pathogens

1. Replication of Double-Stranded RNA Virus;Subviral Pathogens

2. Family Reoviridae • “respiratory, enteric, orphan” • dsRNA • Double icosadehral capsid, 60 nm • Outer capsid + short spikes • Inner nucleocapsid core • Infects plants, insects, animals

3. Genus: Orthoreovirus • Infects avian, mice, humans • Humans – mild URT, GI disease • Fecal-oral route of transmission

4. Genus: Rotavirus • “wheel-like spokes” • Infect animals, humans • Fecal-oral route, respiratory secretions • Infantile diarrhea, gastroenteritis; <5 years of age • USA – winter epidemics; less now due to vaccination • Worldwide epidemics; developing countries >1 million infant deaths/year

5. Genus: Coltivirus • Colorado tick fever virus • Transmission by tick bite to animals & humans • Fever, headache, severe myalgia • May lead to meningitis, encephalitis

6. Human Reovirus: dsRNA Genome • Ten dsRNA segments (L, M, S) • Total genome = 23.5 kb • S1 mRNA has: • two overlapping translational reading frames with alternate initiation site • translates for two proteins • Encodes for eleven viral proteins (λ, μ, σ)

7. Reovirus: Outer Capsid Proteins • σ1 dimer - hemagglutinin • Attachment to cell receptor • Inhibits cell DNA synthesis • μ1C – activates viral RNA pol • σ3 – inhibits cell RNA / protein synthesis

8. Reovirus: Core Proteins • Enzymes for RNA synthesis • λ1/σ2 complex (polymerase) • λ2 (capping enzyme) • λ3 (polymerase)

9. Reovirus: Entry / Partial Uncoating • Receptor mediated endocytosis • Lysosomal fusion results in outer capsid degraded • Release of infectious subviral core particle into cytoplasm • dsRNA • core enzymes (λ1/σ2, λ2, λ3) • RNA pol activated by uncoated outer capsid protein (μ1C)

10. Reovirus: Conservative mRNA Transcription • Occurs within intact subviral core particle in cytoplasm • dsRNA unwinds (viral helicase) • mRNA copied from (-)RNA strand • Daughter mRNAs exit through vertices into cytoplasm • Parent dsRNA remains in subviral core particle

11. Rotavirus Particles: mRNA Release

12. Reovirus: mRNA Translation • Once in cytoplasm, immediate mRNA translation on ribosomes • Regulated viral gene expression: • Four “early” mRNAs code for nonstructual proteins • Six “late” mRNAs code for nonstructual and structual proteins

13. Reovirus: Genome Replication • “late” structual proteins assemble into developing inner core • Ten viral mRNA gene segments inserted into inner core • Copying of (-)RNA strand on viral mRNA to make dsRNA genome • New inner core used for: • mRNA transcription • progeny virions

14. Reovirus: Assembly and Release • Outer capsid forms around inner core into double capsid • Release of virions by cell lysis

15. Subviral Pathogens • Hepatitis delta virus (HDV) – requires a “helper virus” • Viroids – very small infectious RNA • Prions –proteineous infectious particle

16. Hepatitis Delta Virus (HDV) • Envelope from HBV (3 surface gp) • (-)RNA genome complexed with viral protein (delta antigen) • ~15 million infected worldwide • ~40% of fulminant hepatitis infections

17. HDV: (-)RNA Genome • Circular, rod shape due to base pairing, 1.7 kb • Similar to viroids • Defective virus • Replication requires hepatitis B virus (HBV) that supplies replicative functions & viral envelope

18. HDV RNA Synthesis • Entry, uncoating, (-)RNA genome & associated delta antigen transported to nucleus • Viral (+)antigenome RNA synthesis by cell RNA polymerase II • Subgenomic mRNA by two mechanisms: • by interrupted antigenome transcription • by autocatalytic ribozyme activity of circular RNA to linear mRNA

19. HDV Disease • Transmitted by blood, body secretions; similar to HBV, HCV • Two types infection: • Coinfection with HBV • Superinfection (“upon”) chronic HBV patient • Possible chronic disease - increases risk for liver damage and cancer

20. Potato Spindle Tuber Viroid • Small single strand infectious (-)RNA, circular genome, self-complementary (forms dsRNA rod structure) • Genomes of 250-360 nucleotides • Capable of autonomous replication • Appear to encode no proteins • Genomes all contain 5 regions called domains

21. Viroid Genome Replication • Use of cell RNA polymerase II • Double strand helical arrangement of viroid RNA competes effectively with cell DNA for RNA pol II • Cell RNA polymerase I may also play a role

22. Viroid Disease • Transmitted plant to plant: • Mechanical damage • Insects • Seeds, cuttings • Potato spindle tuber viroid • Chrysanthemum stunt viroid • Destroy important crops

23. Viroid Pathogenesis • P domain complementary to cell 7S-RNA (involved in protein translocation) • Postulate that viroid-7S RNA hybrids disturb proper transport of cell proteins • Leads to alteration in plasma membrane structure seen in viroid infections

24. Prions • No nucleic acid; infectivity not inactivated by nucleases • Infectious proteins (PrP); destroyed by proteases • Long incubation period (up to 30 years) • Formerly termed “unconventional slow viruses” • Test by proteinase K digestion, Western Blot analysis of PrP protein: • PrPC – cell protein, destroyed by PK • PrPCJD – prion protein, resistant to PK

25. Prion Protein (PrP) • PrP are 27-30 kd • A cellular protein with unusual folding pattern • In EM, PrPres (from patient) appears as large macromolecular fibrils • Interferes with neuron cell function

26. Prion Diseases • Spongiform encephalopathies in mammals • Sheep – scrapie • Cattle – bovine spongiform encephalopathy (BSE); mad cow disease • Humans – Kuru (“shivering”, New Guinea), Cretzfeldt-Jacob disease (CJD)

27. Viral Evolution • Three theories on the origin of subcellular entities: • Regressive Model • Cellular Constituent Model • Prebiotic RNA Model

28. Regressive Model • Degenerate progeny of other obligate intracellular parasites • Dispense with all but a few genes • Rely entirely upon host cell for metabolic requirements

29. Regression of Bacteria to Viruses

30. Cellular Constituent Model • Descended from normal cellular DNA or RNA • Developed the ability to replicate autonomously • Acquired an origin of replication, replicase, gene(s) for protein capsid

31. Prebiotic RNA Model • First genetic material to develop was RNA • Descendents of self-replicating prebiotic RNA molecules • Became parasites within true cells

32. Theories of Viral Origin

33. Life on the Edge • “A virus is a virus!” • “Whether or not viruses should be regarded as organisms is a matter of taste.” • French Nobel laureate Andre Lwoff, 1959, 1962

34. Life on the Edge • “The very essence of the virus is its fundamental entanglement with the genetic and metabolic machinery of the host.” • American Nobel laureate Joshua Lederberg, 1993

35. Life on the Edge • “It takes a genome. How a clash between our genes and human life is making us sick.” • Greg C. Gibson, Ph.D.; Center for Integrative Genomics, School of Biology, Georgia Institute of Technology; 2010

36. Reading & Questions • Chapter 15: Replication Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression

37. QUESTIONS???

38. Class Discussion – Lecture 8 • 1. How are various Reovirus “structural particles” used for its mRNA transcription and dsRNA replication? • 2. Is Hepatitis delta virus (HDV) dependent on a host cell RNA polymerase for its transcription and replication? • 3. Why are Prions described as “self-replicating” entities?

39. MICR 401 SECOND EXAM • Thursday, Nov. 8, 2012 • Rhabdovirus thru Prions + Life on the Edge • Lecture and Reading • Case Study #1-8 • Objective questions (MC, T/F, ID) • Short essay questions (similar to Class Discussion,Text chapter, Case Study questions)