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dsRNA viruses

dsRNA viruses. Particles typically do not have envelopes Segmented genomes are common; genomes may or may not be divided into in separate particles Transcription is often associated with intact particles Typically cytoplasmic replication

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dsRNA viruses

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  1. dsRNA viruses • Particles typically do not have envelopes • Segmented genomes are common; genomes may or may not be divided into in separate particles • Transcription is often associated with intact particles • Typically cytoplasmic replication • Many viruses of fungi and lower eukaryotes in this group

  2. Families of dsRNA viruses • Reoviridae – largest family, 10-12 segments, infect mammals, invertebrates, plants & fungi • Partitiviridae – 2 or 3 segments, genetically simple viruses, infect fungi and plants • Chrysoviridae – 4 segments, infect fungi • Totiviridae – 1 or 2 segments, infect fungi and lower eukaryotes • Cystoviridae – 3 segments, enveloped virions, infect bacteria • Birnaviridae – 2 or 3 segments, infect vertebrates, invertebrates

  3. TotiviridaeYeast “killer” viruses • Members of the family Totiviridae • Do not cause disease in infected cells • May contain 1 (non-killer) or 2 (killer) segments of dsRNA, in separate particles • Segment 1 (L or L-A segment) contains information required for replication and packaging; can replicate alone • Segment 2 (M, M1, M2, etc.) if present contains gene for yeast-specific toxin, and gene for immunity to that toxin; requires Segment 1 for replication and packaging

  4. Yeast “killer” viruses • Replication of killer viruses has been studied extensively • Yeast provides excellent genetic model for studying virus replication • No easy infectivity system for yeast – particles infect only with difficulty • No reverse genetics system available, despite extensive effort

  5. “Killer” viruses produce toxins that affect only yeast strains that are closely related to their own host strain. They increase the ecological fitness of their host, and thus enhance chances for their own survival. Killer virus + and – strains of Saccharomyces cerevisiae streaked onto a lawn of susceptible S. cerevisiae. Killer toxin secreted from K+ cells results in clearing zone of dead cells in lawn. (Left) Cryo-electron microscopic reconstruction of Saccharomyces cerevisiae virus L-A (ScV-L-A) at 16  Å resolution (Caston et al.). The view shown is along a 5-fold axis of the icosahedral particles. (Right) Negative contrast electron micrograph of Helminthosporium victoriae virus 190S (HvV-190S) virions, a representative species in the genus Totivirus. The bar represents 50  nm. From Wickner chapter in Knipe 2000, Fund. Virol (Lippencott)

  6. Yeast L-A virus particles contain many copies of the coat protein (gag) one or two copies of the gag-pol fusion protein. The large dsRNA is encapsidated one copy per particle; the satellite RNA is encapsidated separately until a “headful” is reached. From Wickner chapter in Knipe 2000, Fund. Virol (Lippencott)

  7. Yeast virus RNA1 encodes the coat protein (gag) and RNA polymerase (pol). It alone represents the complete viral genome. From Wickner chapter in Knipe 2000, Fund. Virol (Lippencott)

  8. Another view of the yeast L-A virus genome, a typical totivirus Figure 2 Genome organization of Saccharomyces cerevisiae virus L-A (ScV-L-A). The virion-associated RNA polymerase catalyzes in vitro end-to-end transcription of dsRNA by a conservative mechanism to produce mRNA for capsid proteins. In the case of ScV-L-A, all of the positive strand transcripts are extruded from the particles. The positive strand of satellite RNA M1, or deletion mutants of L-A or M1, on the other hand, often remain within the particle where they are replicated to give two or more dsRNA molecules per particle (headful replication). The positive ssRNA of ScV-L-A is the species encapsidated to form progeny virus particles. The encapsidation signal on ScV-L-A or M1 positive sense ssRNA is a 24 b stem-loop sequence located 400  nts from the 3  -end in each case. The Gag protein must be acetylated (by the cellular Mak3p) for assembly and packaging to proceed. These particles have a replicase activity that synthesizes the negative strand on the positive strand template to produce dsRNA, thus completing the replication cycle. Replication requires an internal site overlapping with the packaging signal, and a specific 3  -end sequence and secondary/tertiary structure. Virions accumulate in the cytoplasm.

  9. Yeast killer toxin and immunity proteins are both encoded from the satellite RNA2. Structure and expression are very similar to insulin.

  10. Reovirus properties • 10-12 dsRNA segments (9 segment reovirus recently identified, not included here) • Found in mammals, invertebrates, plants • Terminal sequences conserved, but slightly different for each segment • Particle important for replication, capping, mRNA production • No known nuclear component to infection cycle – all cytoplasmic • Very important for early RNA studies • Discovery of capping (Aaron Shatkin) • Studies on translation initiation (Marilyn Kozak)

  11. Reovirus diseases • REO- Respiratory Enteric Orphan virus • Human reoviruses associated with respiratory diseases, especially in infants • Rotavirus is an important cause of diarrhea in young children • Bluetongue virus causes important disease of cattle • Plant reoviruses very important in grasses • Rice dwarf virus was the first virus shown to be vectored • Wound tumor virus was first shown to replicate in vector and led to discovery of sucrose gradients; not an important pathogen • Fungal reoviruses identified relatively recently

  12. Family Reoviridae

  13. Structural and nonstructural proteins encoded by Mammalian reovirus 1

  14. Reovirus particle morphology • Complex 2 or 3 shelled particles • No lipid envelope • Particle comprised of 8 or more proteins • Well-studied T=13 structure • May or may not have surface projections • 50 nanometer core is transcriptionally active

  15. Reovirus genome • 10-12 segments dsRNA • Packaged 1 copy per particle • 22-28 kb total genome size (0.8-4.5 kb each segment) • Transcripts represent genome-length mRNAs • Most genome segments monocistronic, some bicistronic or tricistronic • Genome segments can reassort between related strains • Short 5’ and 3’ non-translated regions

  16. Cryphonectia viruses and transposons AC-like transposon Mitovirus Reovirus Pot-like transposon Hypovirus mtDNA Mitochondrion nucDNA Chrysovirus Nucleus Partitivirus Cytoplasm

  17. Virion ISVP ISVP Core Virion Infectious Subvirion Particles (ISVPs) Core Transcriptionally active Dryden, K.A., G. Wang, M. Yeager, M.L. Nibert, K.M. Coombs, D.B. Furlong, B.N. Fields, and T.S. Baker. 1993. J. Cell Biol.122:1023-1041.

  18. Reovirus “spider” – electron micrograph showing one end of each dsRNA molecule attached to disrupted viral core.

  19. Mammalian orthoreovirus 3 structure and genome organization Infectious subviral particle (ISVP) Electron micrograph Virion Core One copy of each dsRNA per particle Outer capsid Core turret Outer capsid Non-struct. Non-struct. Outer capsid Core Core Core Core Subcellular localization RdRp Attachment Helicase NTPase Membrane penetration Methyltransferase, guanylyltransferase Assembly? Modified from Flint et al., Principles of Virology 2nd Ed., ASM Press

  20. Reoviruses replicate in the cytosol of infected cells. Following penetration of the cellular membrane, viral cores begin transcribing the 10 viral genome segments. The 10 viral genome segments encode 12 viral proteins (8 structural and 4 nonstructural). The nonstructural protein mNS forms the matrix of viral factories where new cores assemble and begin secondary rounds of transcription. The viral cores are coated with the outer capsid proteins m1, s3, and s1 to form intact virions that are released following cell lysis. http://instruct1.cit.cornell.edu/research/parker_lab/Reovirus.htm

  21. Reoviruses contain exactly one segment of each of the 10-12 segments of dsRNA that constitute the viral genome, encapsidated in a single complex virus particle comprised of 6-8 proteins dsRNA 1 dsRNA 2 dsRNA 3 dsRNA 4 dsRNA 5 dsRNA 6 dsRNA 7 dsRNA 8 dsRNA 9 dsRNA 10 Modified From Alan Cann by BIH

  22. mRNAs are likely transcribed at the transcription complexes at each of 12 vertices of the icosahedral particles Modified From Alan Cann by BIH

  23. Capped, methylated mRNAs are transcribed by reovirus core particles Modified From Alan Cann by BIH

  24. Plant Reoviruses consist of Three Distinct Genera • Three major genera differ in 5’ and 3’ termini and in protein coding sequences Members of the genus have 10 or 12 dsRNAs. • Elicit tumors arising from abnormal phloem development. • Transmitted by leafhoppers or plant hoppers. • Viruses multiply in their vectors. Fiji Disease Virus Tumor Reovirus phylogenetic tree

  25. Plant Reovirus Genera Leafhopper vector Susceptible and Resistant Sugarcane cultivars. • ORYZAVIRUS GENUS • Ten dsRNA segments • Leafhopper transmitted. • Infects grasses and • causes galls on leaves. • Rice ragged stunt virus • is type member. FIJIVIRUS GENUS Ten dsRNA segments. Infect Grasses & cause leaf & stem galls. Planthopper transmitted. Fiji disease virus is the type member, several other members known. PHYTOREOVIRUS GENUS Twelve dsRNA segments. Infect Dicots & Grasses. Cause tumors on dicot roots & wounded stems. Rough galls appear on cereal leaves. Leafhopper transmitted. Wound tumor virus is the type member. WTV was first reovirus discovered. Clover tumors on roots.

  26. Reoviruses package exactly one copy of each segment per particle Wound tumor virus Deletion mutants of WTV that lack complete segments required for leafhopper transmission can be generated by serial passage in plant host. Deleted segments are packaged and replicated with the same efficiency as parent segment. When + strand RNA sequence of WTV is folded in silico with an RNA folding program, terminal sequences are shown to be inverted terminal repeats. Terminal sequences of defective (deletion) segments of WTV mutants are the same as the terminal sequences of the parent segment. From Anzola et al., PNAS 1987

  27. Photo Credit: F.P. Williams, U.S. EPA Rotavirus

  28. Rotaviruses differ in structural details from orthoreoviruses, but major features are similar across all reovirus genera

  29. Rotaviruses are Major Causes of Diarrhea L S 1 1 2,3 2,3 4 4 VP4 5 VP7 5 6 6 VP2 7,8,9 7,8,9 dsRNA VP6 10 Aqueous Channel 11 10 11 Rotavirus Genus 11 dsRNA segments.Wheel-like capsids with smooth outer virion. Extremely serious gastroenteritis in almost all animal species. Three main strains in humans. First discovered in 1973 in humans. Fecal-Oral transmission. 125 million severe diarrhea cases world-wide annually. Twenty-Five Million Clinic Visits. Two Million hospitalized with severe illness. Up to 600,000 deaths mostly among malnourished infants. As high as 30% deaths. One to two day Incubation One week Illness Must rehydrate sick children. Strain variation of rotavirus dsRNAs. Rotavirus ranks sixth among the worlds global killers. Location of proteins & dsRNAs.

  30. Diarrhea Causing Agents in World Rotavirus-induced disease incidence is about the same in developed and developing countries; good sanitation does not reduce incidence.

  31. But annual Rotavirus diarrhea deaths are much greater in developing countries

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