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

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

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

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

(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.

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.

“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.

From Wickner chapter in Knipe 2000, Fund. Virol (Lippencott)

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)

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)

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.

Another view of the yeast L-A virus genome, a typical totivirus

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

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)

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

Genus Segments Host VectorOrthoreovirus 10 Mammals None

Orbivirus 11 Mammals Mosquitoes, fliesRotavirus 11 Mammals NoneColtivirus 12 Mammals Ticks

Seadornavirus 12 Mammals TicksAquareovirus 11 Fish NoneIdnoreovirus 10 Mammals None Cypovirus 10 Insect NoneFijivirus 10 Plant Planthopper

Phytoreovirus 12 Plant LeafhopperOryzavirus 10 Plant Planthopper

Mycoreovirus 11 or 12 Fungi None?

Family Reoviridae

Structural and nonstructural proteins encoded by Mammalian reovirus 1

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

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

Nucleus

MitochondrionnucDNA

mtDNA

Cytoplasm

ReovirusMitovirusAC-like transposon

Pot-like transposon

Hypovirus

Chrysovirus

Partitivirus

Cryphonectia viruses and transposons

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.

Infectious SubvirionParticles (ISVPs)

Virion

Core

Transcriptionally active

Virion ISVPISVP Core

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

Modified from Flint et al., Principles of Virology 2nd Ed., ASM Press

Mammalian orthoreovirus 3 structure and genome organization

Virion Infectious subviral particle (ISVP) Core

Electron micrograph

RdRpMethyltransferase,guanylyltransferase

Helicase NTPase

CoreCore turret

Core CoreOuter capsid

Non-struct.

Non-struct.

Outer capsid

CoreOuter capsid

Membrane penetration

AttachmentAssembly?

Subcellular localization

One copy of each dsRNA per particle

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 NS 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 1, 3, and 1 to form intact virions that are released following cell lysis. http://instruct1.cit.cornell.edu/research/parker_lab/Reovirus.htm

Modified From Alan Cann by BIH

dsRNA 1dsRNA 2dsRNA 3

dsRNA 4

dsRNA 5dsRNA 6

dsRNA 7dsRNA 8dsRNA 9

dsRNA 10

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

mRNAs are likely transcribed at the transcription complexes at each of 12 vertices of the icosahedral particles

Modified From Alan Cann by BIH

Capped, methylated mRNAs are transcribed by reovirus core particles

Modified From Alan Cann by BIH

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.

Fiji Disease Virus Tumor Reovirus phylogenetic tree

Elicit tumors arising from abnormal phloem

development.Transmitted by leafhoppers or plant hoppers.Viruses multiply in their vectors.

Plant Reovirus GeneraFIJIVIRUS 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.

ORYZAVIRUS GENUS Ten dsRNA segments Leafhopper transmitted. Infects grasses and causes galls on leaves. Rice ragged stunt virus is type member.

Leafhopper vector

Susceptible and ResistantSugarcane cultivars.

Clover tumors on

roots.

Reoviruses package exactly one copy of each segment per particle

When + strand RNA sequence of WTV is folded in silico with an RNA folding program, terminal sequences are shown to be inverted terminal repeats.

From Anzola et al., PNAS 1987

Wound tumor virus

Terminal sequences of defective (deletion) segments of WTV mutants are the same as the terminal sequences of the parent segment.

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.

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

Rotavirus

Rotaviruses differ in structural details from orthoreoviruses,

but major features are similar across all reovirus genera

Rotavirus Genus 11 dsRNA segments.Wheel-

like capsids with smooth outer virion.

Extremely serious gastroenteritisin 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.

VP4

VP2

VP6

VP7

Aqueous Channel

dsRNA

Rotaviruses are Major Causes of Diarrhea1

2,34

56

7,8,9

1011

12,34

5

6

7,8,9

1011

S L

Strain variation of rotavirus dsRNAs.

Location of proteins & dsRNAs.

Rotavirus ranks sixth among the worlds global killers.

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

Diarrhea Causing Agents in World

But annual Rotavirus diarrhea deaths are much greater in developing countries