Replication of Double- Stranded RNA Virus; Subviral Pathogens

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

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

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

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

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)

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

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

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)

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

Rotavirus Particles: mRNA Release

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

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

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

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

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

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

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

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

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

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

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

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

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: –PrP C – cell protein, destroyed by PK –PrP CJD – prion protein, resistant to PK

Prion Protein (PrP) PrP are kd A cellular protein with unusual folding pattern In EM, PrP res (from patient) appears as large macromolecular fibrils Interferes with neuron cell function

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)

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

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

Regression of Bacteria to Viruses

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

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

Theories of Viral Origin

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

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

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

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

QUESTIONS???

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?

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)