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Ds RNA PLANT VIRUSES

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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

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• 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.

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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.

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• 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%.

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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.

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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.

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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).

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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 REPLICATION1. 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.

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3. Genus Phytoreovirus• 12 genome segments, RDV. 70 nm. 1066 to 4423 bp long. • Ten of the genome segments have one ORE one (4) has two ORFs and one

(8) 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.

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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.

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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.

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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.

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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

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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)

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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)

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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.

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The end

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.

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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

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•Part 2

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Core-Associated Genome ReplicationMechanisms 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

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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

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• 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 triggersthe 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 the same time as the core-associated viral enzymes convert them into dsRNA.

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• 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.

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• 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.

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• 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)

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• 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

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• 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 .

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• 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.

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

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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.

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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.

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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.

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• 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.

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)

The priming loop (blue)

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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

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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.

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• 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

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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

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• 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-CUCUCUCUCU-3 and templates lacking this 3-sequence are packaged, but not replicated, demonstrating that an additional level of specificity occurs during dsRNA synthesis

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• 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.

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• 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

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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?

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• 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

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• 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.

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Reovirus: dsRNA Virus Strategy

Subviral particles in cytoplasm are sites of RNA synthesis

Proteolysis during entry through lysosome activates RNA synthesis

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

‘core’

Capped ss(+) RNA [mRNAs] synthesized

[10 dsRNA genome segments]

RdRP: m1 packaged in virion

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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

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

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