Replication of Double-Replication of Double-Stranded RNA Virus;Stranded RNA Virus;Subviral PathogensSubviral Pathogens

Replication of Double-Replication of Double-Stranded RNA Virus;Stranded RNA Virus;Subviral PathogensSubviral 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 250-360 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: – PrPC

– cell protein, destroyed by PK– PrPCJD – prion protein, resistant to PK

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

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)