Transcript 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
• 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
Totiviridae
Yeast “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
“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)
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)
Another view of the yeast L-A virus genome, a typical to
Figure 2 Genome organization of Saccharomyces cerevisiae virus L-A (ScV-L-A). The virionassociated 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 ScVL-A is the species encapsidated to form progeny virus particles. The encapsidation signal on ScV-LA 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.
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
Family Reoviridae
Genus
Orthoreovirus
Orbivirus
Rotavirus
Coltivirus
Seadornavirus
Aquareovirus
Idnoreovirus
Cypovirus
Fijivirus
Phytoreovirus
Oryzavirus
Mycoreovirus
Segments
10
11
11
12
12
11
10
10
10
12
10
11 or 12
Host
Mammals
Mammals
Mammals
Mammals
Mammals
Fish
Mammals
Insect
Plant
Plant
Plant
Fungi
Vector
None
Mosquitoes, flies
None
Ticks
Ticks
None
None
None
Planthopper
Leafhopper
Planthopper
None?
Structural and nonstructural proteins encoded by Mammalian re
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
Cryphonectia viruses and transposons
AC-like transposon
Pot-like transposon
Mitovirus
Hypovirus
mtDNA
nucDNA
Reovirus
Mitochondrion
Chrysovirus
Nucleus
Cytoplasm
Partitivirus
Virion
Virion
Infectious Subvirion
Particles (ISVPs)
Core
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.
Transcriptionally
active
ISVP ISVP
Core
Reovirus “spider” – electron
micrograph showing one end of
each dsRNA molecule attached
to disrupted viral core.
Mammalian orthoreovirus 3 structure and genome organization
Electron micrograph
Infectious subviral
particle (ISVP)
Virion
Core
One copy of each
dsRNA per particle
Core
Core
turret
Core
RdRp
Methyltransferase,
guanylyltransferase
Helicase
Core
NTPase
Outer
capsid
Nonstruct.
Membrane
penetration
Outer
capsid
Core Non- Outer
struct. capsid
Attachment
Assembly?
Subcellular
localization
Modified from Flint et al., Principles of Virology 2nd Ed., ASM Press
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
dsRNA 1
dsRNA 2
dsRNA 3
dsRNA 4
dsRNA 5
dsRNA 6
dsRNA 7
dsRNA 8
dsRNA 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
Modified From Alan Cann by BIH
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.
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
Plant Reovirus Genera
ORYZAVIRUS GENUS
Ten dsRNA segments
Leafhopper transmitted.
Infects grasses and
causes galls on leaves.
FIJIVIRUS GENUS
Ten dsRNA segments.
Infect Grasses & cause
leaf & stem galls.
Planthopper transmitted.
Fiji disease virus is the
type member, several
other members known.
Susceptible and Resistant
Sugarcane cultivars.
Leafhopper vector
Clover tumors
on roots.
Rice ragged stunt virus
is type member.
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.
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
Rotavirus
Photo Credit:
F.P. Williams, U.S. EPA
Rotaviruses differ in structural details from
orthoreoviruses,
but major features are similar across all reovirus
genera
Rotaviruses are Major Causes of Diarrhea
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.
Rotavirus ranks sixth among
the worlds global killers.
Strain variation of
rotavirus dsRNAs.
S L
1
2,3
4
1
2,3
4
5
5
6
6
7,8,9
7,8,9
10
11
10
11
VP4
Aqueous
Channel
VP2
dsRNA
VP6
VP7
Location of proteins & dsRNAs.
Diarrhea Causing Agents in World
Rotavirus-induced disease incidence is about the
same in developed and developing countries;
good sanitation does not reduce incidence.
But annual Rotavirus diarrhea deaths are much
greater in developing countries