Ds RNA PLANT VIRUSES There are three main families of dsRNA plant viruses discussed in the document: Reoviridae, Partitiviridae, and Endornaviridae. Reoviruses have multiple linear dsRNA segments and carry their own transcription and replication enzymes into host cells. Partitiviruses have two dsRNA segments that encode a polymerase and capsid protein. Endornaviruses have a single large dsRNA genome but do not form virions outside the cell. All three families replicate their genomes within a protected protein core using a virion-associated RNA-dependent RNA polymerase.

1. Ds RNA PLANT VIRUSES
1

2. Ds RNA Viruses
• Eight distinct families of dsRNA viruses
• Provide their own transcription (and sometimes capping) enzymes and
carry them into the host cell & do not utilize the cell's replication enzymes
• Consist of multiple linear dsRNA almost monocistronic
2

3. • The internal capsid layers and internal virion-associated enzymes,
which are the most conserved viral proteins, In contrast, it is the
outer capsid layers (and some non-structural proteins), which appear
to be adapted for virus transmission.
• The outer capsid layers (120 copies of a large, approximately
triangular, protein) of the icosahedral viruses are in each case
responsible for initiating infection by delivering the transcriptionally
active core of the virion into the cytoplasm of the host cell.
3

4. Family Reoviridae
• The genomes of plant reoviruses (Reo: from Respiratory
enteric orphan) comprise either 10 or 12 RNA segments.
• transcriptase that uses the RNA in the particle as template
to produce ssRNA copies
• Four types of gene products are recognized, those that
make up the capsid (structural proteins), those involved
with RNA replication, non-structural proteins, and those
for which no function is known.
• The plant-infecting reoviruses replicate in their insect
vectors and are not considered to be seed transmitted.
4

5. • Transovarial (vertical) transmission of reoviruses in insect
vectors has been demonstrated for the fijiviruses Fiji disease
virus (FDV), Oat sterile dwarf virus (OSDV), Maize rough
dwarf virus (MRDV), and Nilaparvata lugens virus (NLV), and
the phytoreoviruses Rice dwarf virus (RDV), Wound tumor
virus (WTV) and Rice gall dwarf virus (RGDV).
• Phytoreoviruses are transmitted at higher rates (1.8%–100%)
than fijiviruses (0.2%–17%). Although the rate of transmission
is low for fijiviruses, FDV was transmitted transovarially for
several generations and transmissivity was maintained for 6
years at 100%.
5

6. 6

7. 1. Genus Fijivirus
• 10 RNA segments. 65-70 nm. Hypertrophy and enations in their
graminaceous hosts, and are transmitted by delphacid
planthopppers. (subfamily: Spinareovirinae)
• Fijivirus particles are double-shelled with 'A„- type spikes on the 12
vertices of the outer shell and 'B'-type spikes on the inner shell or core.
• The gene product from segment 3 of NLRV the major core protein and
those from segments 1 and 7 contribute enzymatic activities also found in
the core. The product from segment 2 has been attributed to the 'B' spike
on the inner core.
7

8. Fijivirus
• 10 segments coding for 12 proteins. Segments
size range from 1.4 to 4.5 kb. Genome total size is
about 27-30 kb.
• Type: Fiji disease virus (FDV)
• Main: Maize rough dwarf virus (MRDV)
Mal de Rio Cuarto virus (MRCV)
Rice black streaked dwarf virus (RBSDV)
Oat sterile dwarf virus (OSDV)
Nilaparvata lugens reovirus (NLRV)
Pangola stunt virus (PaSV)
• Group 5 contains NLV, which infects the brown
leafhopper but does not replicate in rice.
The core protein that has NTP-binding activity is encoded by
segment 8 (1927bp) of RBSDV, possibly by segment 7 (1936 bp)
of MRDV, segment 9 (1893 bp) of OSDV and segment 7 (1994
bp) of NLRV; however, segment 9 of OSDV is larger than
segment 8.
8

9. 2. Genus Oryzavirus
• Contains 10 segments coding for 12 proteins. Segments size range
from 1162 to 3849 bp. Genome total size is about 26 kb (RRSV).
• Unlike the other two genera, the RNA polymerase is not encoded
on the largest RNA segment but on the second largest one.
However, like the other two genera it is the largest gene product.
• 78–80 nm. replicate in both their delphacid planthopper vectors
and Poaceae hosts.
• Pentameric turrets (VP2) sit on the outside of the innermost
capsid (VP3). Each turret is surrounded by five peripheral trimers (VP9) and each trimer binds to three clamp
proteins (VP4B).
9

10. Oryzavirus
• The B-type spikes are encoded on segment 1 and the A-type spikes,
which are involved in vector transmission by delphacid planthoppers,
are encoded by segment 9.
• The dsRNA genome is never completely uncoated, to prevent activation
of antiviral state by the cell in response of dsRNA. The viral polymerase
synthesizes a capped mRNA from each dsRNA segment. This capped
mRNA is translocated to the cell cytoplasm where it is translated.
• CYTOPLASMIC REPLICATION
1. Virus penetrates into the cytoplasm.
2. Transcription of the dsRNA genome by viral polymerase occurs inside the
virion, so that dsRNA is never exposed to the cytoplasm. This plus-strand
transcript is used as template for translation.
3. (+)RNAs are encapsidated in virion particle, inside which
they are transcribed to give RNA (-) molecules with which
they become base-paired to produce dsRNA genomes.
4. Mature virions are released presumably following cell death
and associated breakdown of host plasma membrane.
10

11. 3. Genus Phytoreovirus
• 12 genome segments RDV. 70 nm. 1066 to 4423 bp long.
• Ten of the genome segments have one ORE one has two ORFs and one
three.(Sedoreovirinae)
• Segments 2, 3 and 8 encode structural proteins; the outer capsid protein
encoded by segment 2 is essential for vector transmission (Nephotettix
cincticeps). The products of segments 1, 5, 7 and possibly one of the
products of segment 12 are involved with RNA replication, and those of
segments 4, 6,10, 11 and one encoded by segment 12 are classed as non-
structural proteins. The inner P3 and outer P8 capsid proteins.
Each double-layered capsid consists of 260 trimers of the
major outer capsid P8 protein and 60 dimers of the inner
capsid P3 protein.
11

12. Phytoreovirus
• SPECIES Type: Wound tumor virus (WTV)
• Main: Rice dwarf virus (RDV)
Rice gall dwarf virus (RGDV)
Tobacco leaf enation phytoreovirus (TLEP)
• RGDV, WTV, and Tobacco leaf enation virus (TLEV) have a
genomic organization similar to that of RDV, but individual
genome segments and encoded proteins have low sequence
identity.
• The three species in this genus infect both monocot and dicot
plant hosts and are transmitted by cicadellid
leafhopper vectors.
12

13. Phytoreovirus
• In plants, RDV P2 interacts with entkaurene oxidase-like
proteins. These enzymes play a role in gibberellic acid synthesis
in plants and their interaction with P2 is likely to be associated
with symptom expression (e.g., gall formation).
• Viral inclusion bodies comprised of Pns10 form tubular
structures ca. 85 nm in diameter and contain virus in insect host
cells Tubules composed of a nonstructural viral protein and
actin-based filopodia to move into neighboring cells.
• Pns4 is associated with minitubular structures ca. 10 nm in
diameter in viruliferous insects, similar to those formed in
animal cells infected.
13

14. Phytoreovirus
• two hypotheses about the evolution of the phytoreoviruses:
either the virus, insect, and plant hosts coevolved or the viruses
evolved as insect viruses that secondarily adapted to replicating in
plant hosts.
• The viruses replicate to higher titers in insect hosts than in plant hosts,
some of the viruses are transmitted through insect eggs, but none is
transmitted through seed.
• In addition, plants are inefficiently infected by single insects, and
cytopathic effects of virus infection are greater in the plant than in the
insect.
14

15. B. Family Partitiviridae
• Two dsRNA genome segments.
• The two plant genera of this family, the genus Alphacryptovirus
and the genus Betacryptovirus.
• In alphacryptoviruses the larger segment encodes the virion-associated RNA
polymerase and the smaller segment codes for the capsid protein. It is thought
that betacryptoviruses have the same genome arrangement.
Alphacryptovirus
Betacryptovirus
15

16. Alphacryptovirus
• About 30 nm in diameter. Icosahedric symmetry is
presumably T=1 but has not been determined. The two
genomic segments are encapsidated separately.
• 2 segments encoding potentially for 2 proteins.
Segments are about 1.7 and 2.0 kb each, total size about
4 kb.
• Family: Partitiviridae Genus: Alphacryptovirus
• Type: White clover cryptic virus 1 (WCCV-1)
16

17. Betacryptovirus
• about 40 nm in diameter. Icosahedric symmetry is
presumably T=1 but has not been determined. The
two genomic segments are encapsidated separately.
• 2 segments encoding potentially for 2 proteins.
Segments are about 2.1-2.25 kb each, total size about
4 kb.
• Family: Partitiviridae Genus: Betacryptovirus Type:
White clover cryptic virus 2 (WCCV-2)
17

18. C - Endornaviridae
• Endornaviridae viruses do not produce virions, that they are
efficiently transmitted through seed, no horizontal spread has been
observed in the field, no potential vectors have been identified and
none is associated with disease symptoms, except for one
associated with sterility.
• Linear dsRNA genome about 14 kb to 17.6 kb. A site specific
break (nick) is found in the coding strand about 1 to 2 kb from the
5‟ terminus. No true viral particles or structures have been
observed.
• The genome encodes for one
ORF potentially cleaved in
several polypeptides.
18

19. CYTOPLASMIC REPLICATION
• Virus genome is transmitted horizontally through mating
or vertically from mother to daughter cells.
• Transcription-translation of genomic RNA produce viral
RdRp and possibly other proteins.
• Replication occurs in cytoplasmic vesicles. Genomic
(+)RNA is copied into its complementary antigenomic
RNA forming new dsRNA genomes.
19

20. References:
• Peter Mertens. The dsRNA viruses. (2004). Virus Research 101: 3–13.
• Taiyun Wei, Akira Kikuchi, Yusuke Moriyasu, Nobuhiro Suzuki, Takumi
Shimizu, Kyoji Hagiwara, Hongyan Chen, Mami Takahashi, Tamaki
Ichiki-Uehara and Toshihiro Omura. The Spread of Rice Dwarf Virus
among Cells of Its Insect Vector Exploits Virus-Induced Tubular
Structures. (2006). Journal of virology, Vol. 80, No. 17, p. 8593–8602.
• Peter. P. C. Mertens, Houssam Attoui and Dennis H. Bamford. The RNAs
and Proteins of dsRNA Viruses. Updated August 2003, using data
provided by Toshihiro Omura. Available at:
http://www.reoviridae.org/dsRNA_virus_proteins/Phytoreovirus.htm.
• Saskia A. Hogenhout, El-Desouky Ammar, Anna E. Whitfield and
Margaret G. Redinbaugh. Insect Vector Interactions with Persistently
Transmitted Viruses. (2008). Annu. Rev. Phytopathol. 46:327–59
20

21. •Part 2
21

22. Core-Associated Genome Replication
Mechanisms of dsRNA Viruses
• studies of these three virus families (the Reoviridae,
Totiviridae and Cystoviridae families):
• (i) RNA synthesis occurs within a protected core via an
anchored RNA-dependent RNA polymerase (RdRp);
• (ii) genome replication and capsid assembly occur
simultaneously; and
• (iii) cis-acting elements in the viral RNA determine
template specificity
22

23. RNA Synthesis Occurs Inside a Protected Core
• To escape intracellular defense mechanisms by
confining their genomes, throughout the entire
course of infection, within one to three concentric
protein shells
• The innermost protein shell not only houses the
segments of viral genomic dsRNA but also
encases the viral RdRp and other enzymes
necessary for mediating RNA synthesis
23

24. • During the entry of a dsRNA virus into a cell, the outer layers of
the virion are sequentially lost, triggering the enzymes within the
core to begin viral transcription ((+)RNA synthesis) using the
endogenous dsRNA genome as template
• Following transcription, the (+)RNA molecules are extruded from
the virion core and into the host cell cytoplasm where they are
translated into viral proteins
(A) Transcription. Entry of a dsRNA virus into a cell triggers
the enzymes (pink and purple) within the core shell (light blue) to begin (+)RNA synthesis using the endogenous
dsRNA genome (blue spirals) as template. Following transcription, the (+)RNA molecules (black lines) are extruded
from the virion core through channels at the fivefold axes.
(B) Replication. Viral core proteins assemble into intermediate structures, which package (+)RNA molecules at the24
same time as the core-associated viral enzymes convert them into dsRNA.

25. • For Reoviridae, newly synthesized viral proteins accumulate in large
cytoplasmic inclusions where the initial stages of virion particle
assembly occur simultaneously with genome replication (dsRNA
synthesis)
• Particularly, viral core proteins assemble into intermediate structures,
which package (+)RNA molecules at the same time as the core-
associated viral enzymes convert them into dsRNA
• The prototypical member of Totiviridae is L-A virus, a pathogen of the
yeast Saccharomyces cerevisiae. L-A is one of the simplest dsRNA
viruses, having only one genome segment, which is encased by a
single shell made up of the viral coat protein (Gag)
• The viral RdRp (Pol) is expressed from the genome as a Gag–Pol
fusion protein due to a –1 ribosomal frameshift and is incorporated
into L-A particles
25

26. • Pol is required for mediating the concerted replication
and packaging of the viral genome segment while
anchored inside the core; however, Gag alone is sufficient
for particle formation
26

27. • The bacteriophage phi 6 (Φ6): The Φ6 virion is a double
layered nucleocapsid (NC) surrounded by a host cell-derived
lipid envelope, which is embedded with several viral proteins
• Φ6 core consists of four viral proteins: a shell protein (P1), a
nodule-like hexameric NTPase (P4), an assembly cofactor
(P7), and an internally anchored viral RdRp (P2)
27

28. • The virion architecture of Reoviridae family members is
similar to Totiviridae and Cystoviridae. Yet, this family
is more complex due to the increased number of genome
segments and capsid proteins.
• Some Reoviridae genera have turrets composed of the
viral capping enzyme(s) (5-triphosphatase, guanylytransferase,
methyltransferase, etc.) that protrude outward from their core
shells at each fivefold axis
28

29. • The structures of Φ6 P2 and mORV λ3 also show
several hollow tunnels that allow the RNA template,
nucleotides, and divalent cations to access the
catalytic site and to permit the exit of nascent RNA.
Both enzymes have a single nucleotide entry tunnel
on one side as well as a single template entry tunnel
approximately 90 away near the top of the protein .
29

30. • Because Cystoviridae transcription occurs using a semi-
conservative mechanism, Φ6 P2 has a single tunnel for
the exit of a dsRNA product, making it a three-tunneled
RdRp.
• In contrast, members of the Reoviridae family use a fully
conservative mechanism of transcription, meaning that
the RdRp separates the dsRNA product into the nascent
(+)RNA and parental (–)RNA strands prior to their exit.
This separation requires mORV λ3 to have two RNA exit
tunnels, making it a four-tunneled RdRp.
30

31. Locations of tunnels within the Φ6 P2 (A) and mORV λ3(B)
structures.
31

32. semi-conservative Φ6 transcription
• The product of semi-conservative Φ6 transcription is a dsRNA
duplex composed of nascent the (+)RNA strand paired with the
parental (–)RNA strand, which is released from P2 via the single
RNA exit tunnel. This dsRNA molecule is separated again, the
(+)RNA transcript is shuttled out of the core, and the (–)RNA
strand is used as a template for another round of transcription.
32

33. fully conservative mORV transcription
• The parental (+)RNA strand that is “peeled-off” the dsRNA segment
stays inside the core and waits to reanneal with its complementary
(–)RNA strand. the parental (–) RNA strand enters λ3 and is used as a
template for nascent (+)RNA strand synthesis, made initially as a
dsRNA duplex & allowing the parental (–)RNA strand and the
nascent (+)RNA strand to exit the enzyme via individual tunnels.
Following release of the two strands from the enzyme, the parental
(–)RNA strand base pairs with its initial (+)RNA partner to reform the
original dsRNA segment, while the nascent (+)RNA transcript
acquires a 5-cap as it is extruded from the core.
33

34. Replication
• Replication initiation without a primer requires
specific molecular interactions to occur
between the template and incoming
nucleotides in order to keep them correctly
positioned at the RdRp active site, to aid in
forming these stable interactions, many viral
RdRps have a region of the protein that
functions as a “stage” on which an initiation
complex is constructed.
34

35. • For the Cystoviridae member Φ6, the carboxy-terminal plug provides such
a “stage” and is referred to as the initiation platform. During Φ6 RNA
synthesis, the RNA template enters P2 and is stabilized by the plug,
allowing it to base pair with incoming initiatory nucleotides near the active
site. In the course of elongation, the carboxy-terminal plug presumably
moves to allow the dsRNA product to egress from the RdRp active site.
• For mORV, the incoming nucleotides enter λ3 and are stabilized against
the priming loop, which is formed by the residues in the tip of the fingers
and the palm sub-domains. Thus, the priming loop functions as a “stage”
for λ3, allowing the incoming nucleotides to base pair with the RNA
template. Like the Φ6 P2 plug, the λ3 priming loop shifts its location
following initiation of phosphodiester bond formation so as not to block
the elongating dsRNA duplex.
The priming loop (blue)
Incoming initiatory nucleotides (pink) are stabilized by the P2 carboxy-terminal plug
(blue) and a motif-F-like structure (teal), allowing them to base pair with the RNA
template (gold) near the catalytic aspartic acids (red)
35

36. Biochemical Studies of dsRNA Viral
Genome Replication
• In vitro VP1 catalyzes dsRNA synthesis in a manner
connected to core assembly, the precise mechanism by
which VP2 triggers the function of VP1 is unknown, it is
possible that the amino terminus of VP2 forms an internal
platform inside the core at the fivefold axis and on which
the RdRp operates.
• the molar ratio of VP1:VP2 required for maximum dsRNA
synthesis was determined to be 1:10
• the amino terminus of VP2 contains a domain critical for
interactions with VP1, VP3, and RNA
• it remains unclear exactly how VP2 activates VP1 to
initiate dsRNA synthesis
36

37. Genome Replication and Capsid
Assembly Occur Simultaneously
• To protect newly made dsRNA from the host cell antiviral response,
(+)RNA packaging into a core-like intermediate pro-cores, can be made
using recombinant proteins P1, P2, P4, and P7.
• These pro-cores are stimulated to package and replicate the (+)RNA
templates (S+, M+, and L+) by incubation in polyethylene glycol, ADP,
Mg2+, and rNTPs.
• The reaction is consecutive in that S+ is packaged first, followed by M+
and then L+. A hexamer of the NTPase protein (P4) mediates the bulk of
(+)RNA packaging at a single fivefold axis, but a cofactor protein (P7)
enhances the efficiency of this process.
• P4 functions like a molecular motor, powering the entry of the (+)RNA
molecules into the pro-core.
• Only after all three templates are packaged inside the pro-core does
(–)RNA strand synthesis begin.
37

38. • An initial interaction occurs between the RdRp
VP1, the capping enzyme VP3, and a single
(+)RNA template, forming a pre-core RI that lacks
polymerase activity
• A VP2 decamer interacts with a pre-core RI to form
a core RI, which is capable of initiating dsRNA
synthesis.
• the cis-acting packaging signals are located in the
(+)RNAs, but are masked in the dsRNA products.
• the requirement of SA11 VP2 for binding VP1,
VP3, and RNA, and for triggering genome
replication ensures that dsRNAs are not produced
until cores are available for their protection
38

39. Cis-Acting RNA Signals Determine
Template Specificity
• A dsRNA virus must pick the correct viral (+)RNA
molecules from a sea of cellular ones.
• This specificity is attributed to the presence of cis-acting
signals that selectively channel the viral RNAs into the
assembly and replication complexes.
• For L-A, a stem-loop structure (internal site, 400
nucleotides from the 3-end) in the (+)RNA forms the
packaging signal that is recognized by the Pol domain of
the Gag–Pol fusion protein.
• Conversion of this packaged (+)RNA to dsRNA requires
this internal site, as well as Sequences in the 3-end of the
template
39

40. • The Cystoviridae member Φ6 specifically recognizes its
RNA template based on a conserved 18-nt sequence at the
5-end, as well as an upstream pac sequence that is unique in
each segment. The pac sequence of each segment folds into
a distinctive stem-loop structure required for organized
packaging.
• Template RNAs should have the 3-sequence 5-CUCUC
UCUCU-3 and templates lacking this 3-sequence are
packaged, but not replicated, demonstrating that an
additional level of specificity occurs during dsRNA
synthesis
40

41. • the packaging signals of mORV reside at the 5-end of (+)RNAs: to
form a stem-loop structure that might serve as a recognition signal for
core proteins
• cis-acting signals in (+)RNAs that support genome replication for the
Reoviridae family:
• (i) a 3-terminal consensus sequence (3CS) 5-UGUGACC-3 and (ii) a
panhandle structure formed by sequences in the 5- and 3-UTRs the
highly conserved , 3CS is the most important, as a deletion of this
region in the context of a viral (+)RNA template completely abolishes
replication.
• the formation of a panhandle structure, as a result of base pairing
between the 5-UTR and 3-UTR, is important for genome replication
& might also promote the proper assortment of the (+)RNAs during
packaging. 41

42. • VP1 is capable of using (–)RNA templates, which
lack both a 3CS and a panhandle structure, for
multiple rounds of transcription.
• The 3-ends of SA11 (–)RNA strands show a less
conserved sequence of 5-(A/U)6AGCC-3 that is
thought to be recognized by VP1 during transcription,
but with a lower affinity than the 3CS of (+)RNAs
42

43. Unanswered questiones:
• What region(s) within the core shell proteins are important
for interactions with the viral enzymes and RNA? Which
domain(s) of the RdRp directly engage the core shell and/or
other viral enzymes? Do the viral enzymes remain tethered
to the inside of the core shell during all stages of viral RNA
synthesis?What changes occur inside the core following
viral entry/uncoating that trigger transcription? What
changes occur inside the core during RNA packaging that
trigger genome replication? How do segmented dsRNA
viruses package equimolar ratios of genome segments? Is
RNA packaging coordinated by protein– RNA interactions
only or are RNA–RNA interactions among segments
important too? What are the roles of viral nonstructural
proteins during packaging and replication?
43

44. • Chrysoviridae family members are similar to pseudo T = 1
cores, but are composed of 60 protein subunits instead of
dimers, making them classic T = 1 structures, package their
four dsRNA segments in separate core shells.
• Birnaviridae family have single-shelled particles that show
T = 13 icosahedral symmetry and are nearly identical to the
structure of Reoviridae outer virion layers & have a VPg-
like protein linked to the 5-ends of their bisegmented
genome, a feature that is seen in several positive-strand
RNA viruses.
• the Hypoviridae family members have a replication strategy
that is more similar to that of positive-strand RNA viruses
than to other dsRNA viruses
44

45. • It is thought that positive-strand RNA viruses mediate RNA
synthesis in association with vesicular or invaginated
membranes to protect their dsRNA replication intermediates
from detection by the host cell antiviral system.
• Membranous positive-strand viral replication complexes
and dsRNA viral cores can be thought of as functionally
analogous structures. Although these shared features cannot
distinguish divergent from convergent evolution, these
parallels suggest that positive-strand RNA and dsRNA
viruses might have an ancestral linkage.
45

46. Reovirus: dsRNA Virus Strategy
Proteolysis during entry through lysosome activates RNA synthesis
Subviral particles in cytoplasm are sites
of RNA synthesis
Capped ss(+) RNA [mRNAs] synthesized
[10 dsRNA genome segments]
RdRP: m1 packaged in virion
‘core’
Extruded into cytoplasm through
channels in 5-fold axes
Remain in cytoplasm: translated
Packaged into new subviral
particles: templates for –RNA
synthesis to produce new dsRNAs
46

47. References:
• Sarah M. McDonald and John T. Patton. Core-Associated
Genome Replication Mechanisms of dsRNA Viruses. Viral
Genome Replication, C.E. Cameron et al. (eds.). Springer
Science+Business Media, LLC 2009.
• Lawton JA, Estes MK, Prasad BV. Mechanism of genome
transcription in segmented dsRNA viruses. Adv Virus Res.
2000;55:185-229 .
• http://instruct1.cit.cornell.edu/research/parker_lab/Reovirus.htm
47

48. http://instruct1.cit.cornell.edu/research/parker_lab/Reovirus.htm
48

49. When + strand RNA sequence of WTV
is folded in silico with an RNA folding
program, terminal sequences are
shown to be inverted terminal repeats.
49