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)