The dsRNA viruses

Virus Research 101 (2004) 3–13

The dsRNA viruses Peter Mertens Institute for Animal Health, Pirbright Laboratories, Ash Road, Pirbright, Woking GU24 0NF, UK

Abstract The dsRNA viruses represent a large, diverse group of pathogens (affecting a very wide range of host species), several of which are of medical, veterinary or agricultural importance. Many of the icosahedral dsRNA viruses show striking structural and functional similarities that reflect the similar problems that they face replicating their dsRNA genomes while avoiding the dsRNA activated defence mechanisms of their host species. These similarities appear to indicate a common if distant ancestry that is not always evident simply by comparison of nucleotide or amino acid sequences. To facilitate the identification and comparisons of cognate proteins from different species, genera and families of dsRNA viruses, a series of tables were originally constructed for the 7th International Symposium of dsRNA viruses held in Aruba in 2000. These have now been updated and extended (for the 8th Symposium, held in Tuscany 2003) and are available from the dsRNA virus website at www.iah.bbsrc.ac.uk/dsRNA virus proteins/. © 2003 Elsevier B.V. All rights reserved. Keywords: dsRNA viruses; Genomes; Pathogen; Orbivirus; Reovirus; Rotavirus; Chrysovirus; Cystovirus; Varicosavirus; Cypovirus; Totivirus; Partitivirus; Birnavirus; Hypovirus

1. Introduction

2. dsRNA viruses: diversity and taxonomy

The dsRNA viruses represent a very large group of different pathogens, affecting a wide variety of terrestrial and non-terrestrial vertebrates, terrestrial and non-terrestrial invertebrates, plants, fungi, and prokaryotes—truly a remarkable assortment of viruses found in a remarkable variety of econiches. The majority (although not all) of these viruses have icosahedral capsid structures (Table 1) and have several similar problems to overcome during the replication of their dsRNA genomes. This has resulted in many clear similarities, not only in replication strategy but also in shared structural and biochemical properties. Indeed, it is possible to identify individual cognate proteins with similar functions or structures, from even distantly related dsRNA viruses, providing evidence of a common ancestry. These similarities are most obvious in the innermost 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 and initiation of infection in different host systems, that show greatest diversity in both the sequences of their components and/or their structural organisation.

Eight distinct families of dsRNA viruses are currently recognised by the International Committee for the Taxonomy of Viruses (ICTV) (Tables 1 and 2), containing several viruses of major medical, veterinary or agricultural importance. For example, rotaviruses are recognised as a major global cause of infant mortality. Bluetongue (caused by bluetongue virus, the prototype Orbivirus) is a disease of ruminants (particularly affecting sheep) that is currently widespread in southern Europe (>800,000 dead animals) (Mertens and Mellor, 2003). Several of the plant viruses cause diseases of major crop species, including wheat and rice (Mayo, 2000; Mertens et al., 2000). The cypoviruses not only constitute a significant threat to the silk industry but also include several viruses that have potential as biological control agents for specific species of insect pests (Mertens et al., 2000). The birnaviruses include infectious bursal disease virus (a major cause of losses in the poultry industry) and infectious pancreatic necrosis virus, which causes significant losses in the salmon fisheries of North America (Leong et al., 2000). Of the 135 dsRNA virus species already recognised (plus 52 that are tentative or unassigned), the largest number (74, plus 30 tentative or unassigned) are classified within the

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P. Mertens / Virus Research 101 (2004) 3–13

Table 1 The families of dsRNA viruses Family

Number of genome segments

Hypoviridae (Hillman et al., 2000)

1 (unpackaged)

Totiviridae (Wickner et al., 2000) Birnaviridae (Leong et al., 2000) Varicosavirus (genus) (Mayo, 2000) Partitiviridae (Ghabrial et al., 2000)

1 2 2 2

Cystoviridae (Bamford, 2000)

3 (co-packaged, equimolar)

Chrysoviridae (Ghabrial et al., 2003)

4 (packaged separately)

Reoviridae (Mertens et al., 2000)

(packaged singly) (co-packaged) (separately packaged) (separately packaged)

10, 11 or 12 (co-packaged, equimolar)

Type of virus particle

Host

∼50–80 nm diameter, pleomorphic vesicles (no capsid) ∼30–40 nm diameter, icosahedral ∼60 nm diameter icosahedral, single shell ∼18 × 320–360 nm, rod shaped ∼30–40 nm diameter, icosahedral protein capsid ∼85 nm diameter, three layer structure, with an envelope surrounding a two layered icosahedral nucleocapsid ∼30–40 nm diameter icosahedral protein capsid ∼70–90 nm diameter icosahedral (one, two or three layered protein capsid)

Fungi Fungi Fish, insects, birds, molluscs Plants Fungi plants Bacteria (Pseudomonas)

Fungi Insects, plants, fish, reptiles, birds, mammals, arachnids, fungi, arthropods, crustaceans

Table 2 the genera and species of dsRNA viruses Family

Genera

Hypoviridae

Hypovirus

1

2 (+2)

3 (+2)

Totiviridae

Totivirus Giardiavirus Leishmaniavirus

1

4 (+4) 1 (+1) 13

4 (+4) 1 (+1) 13

???

Varicosavirus

2

Partitiviridae

Partitivirus Alphacryptovirus Betacryptovirus

2

Birnaviridae

Aquabirnavirus Avibirnavirus Entomobirnavirus Rotifer birnavirus (unassigned)

2 2 2 2

2 1 1 (+1)

5 17 1 (+1)

Cystoviridae Chrysoviridae

Cystovirus Chrysovirus

3 4

1 (?) 4 (+1)

1 (?) 4 (+1)

Reoviridae

Cypovirus Fijivirus Orbivirusb Orthoreovirus Oryzavirus Proposed new genus of insect reoviruses Aquareovirus Rotavirusc Coltivirus Phytoreovirus Seadornavirus Mycoreovirus Unassigned viruses

16 (+3) 8 20 (+12) 4 2 2 (+7) 6 6 (+2) 2 3 (+1) 3 3 (5)

16 (+3)a 8 157 (+12) 6 4 2 (+7) 23 (+5) >23 5 (+1) 3 (+1) 7 (+15) 3 (5)

135 (+52)

337 (+71)

Total

Number of distinct genome segments

10 10 10 10 10 10 11 11 12 12 12 11 or 12 10, 11 or 12

Number of member species (+ tentative or unassigned isolates)

1 14 (+3) 16 (+10) 4 (+1)

Total number of types (serotypes) (+ tentative or unassigned isolates)

1 14 (+3) 16 (+10) 4 (+1)

Compiled using data from Virus Taxonomy: Seventh Report of the International Committee for the Taxonomy of Viruses (with updates). a A large number of cypovirus isolates (>230) have been recovered from a range of different insect host species. Although many of these are related viruses and can be grouped within the 16 Cypovirus species currently recognised, many of them have not been fully characterised and remain unassigned. b The serological relationships between different isolates of some distinct Orbivirus species has not been fully explored. c The identity of Rotavirus serotypes has not been fully explored. In Rotavirus A there are 23 distinct genotypes, although two proteins (VP7 and VP4) are involved in neutralisation, giving rise to 15 ‘G’ serotypes and 14 ‘P’ serotypes (with subtypes in three of these). No information is available for serotypes of the other five confirmed and two tentative species.

P. Mertens / Virus Research 101 (2004) 3–13

family Reoviridae (Mertens et al., 2000), which is by far the largest of the seven virus families. The Reoviridae contains a total of 11 distinct genera (Table 2) that have been recognised by the International Committee for the Taxonomy of Viruses, with an additional genus already proposed and several other viruses that are still unassigned (Table 2). Within the Reoviridae itself the largest number of these viruses (20 species plus 12 tentative species, containing a total of 169 types or serotypes) are assigned to the genus Orbivirus (the orbiviruses) (Mertens et al., 2000). Despite similarities in replication strategy, some clear similarities in virus structure and even the identification of cognate proteins, the dsRNA viruses represent a very diverse group. The level of amino acid and nucleotide sequence similarity between different genera is usually low and often approaches that of random sequence comparisons. For example, even the highly conserved polymerases of several distinct genera within the family Reoviridae show only approximately 10–20% amino acid identity. Indeed in many cases it is difficult to determine the function of the viral proteins by sequence comparisons alone, between these groups. Even within a single genus there can be high levels of sequence difference between species, for example the polyhedrin genes of individual species of Cypovirus show approximately 60% nucleotide difference, and many other genera show 40–50% nucleotide difference in cognate genes. Perhaps the most extreme example of divergence within a single genus is the rotaviruses, with >75% amino acid divergence between the polymerase of rotavirus A and B. With these levels of sequence diversity and the wide variety of host species that are targeted by these viruses, it may be time to consider recognition of a taxonomic ‘order’ of dsRNA viruses.

3. Common solutions to a common problems In order to infect and successfully replicate in their target host species, the dsRNA viruses have had to overcome a number of specific biochemical problems. Although very stable (as demonstrated by the retention of virus particle infectivity over long periods even at room temperature), dsRNA molecules are ineffective as mRNA templates for translation and cannot themselves function as templates for host cell transcriptases. These viruses must therefore provide their own transcription (and sometimes capping) enzymes and carry them into the host cell with them, allowing them to synthesise the functional mRNAs required to initiate viral protein synthesis. However, many host cells also contain antiviral defence mechanisms (Jacobs and Langland, 1996) including induction of apoptosis, interferon production, modification of host cell translation mechanisms and even RNA silencing (Gitlin and Andino, 2003; Goldbach et al., 2003) that specifically recognise and would be activated by naked dsRNA within the host cell cytoplasm (Jacobs and Langland, 1996). In-

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deed the ubiquitous nature of these defences suggests that the dsRNA viruses may themselves represent an ancient lineage that has diversified, along with their host species, from biologically ancient common ancestors. In order to avoid exposure to the host cell cytoplasm, many of the dsRNA viruses retain their genomes, and mRNA synthesising enzymes, within stable closed protein capsid shells. These ‘nano’ transcription machines are in effect the basic infectious unit of the virus that must be delivered intact into the host cell cytoplasm in order to initiate the processes of replication. The similar role of these structures (and the common problems faced by the virus in their assembly) is reflected in remarkable similarities that exist in both the organisation and structure of the innermost capsid layer and internal enzymes. These similarities are evident not only for many of the members of the Reoviridae but even between some members of the more distant families of dsRNA viruses (Table 3). One of the best examples of this functional equivalence, suggesting an ancestral relationship, is provided by a structural comparisons of the inner capsid layer of several of the icosahedral viruses. In each case the innermost capsid shell is composed of 120 copies of a large, approximately triangular, protein (P1 of phi 6 (Table 4), lambda 1 of reovirus (Table 5), VP3 of BTV (Table 6), VP2 of rotavirus (Table 7) and the capsid protein of Saccharomyces cerevisiae LA virus (Table 9)). This protein provides an apparently simple yet elegant mechanism of assembling the inner icosahedral capsid shell, which has alternatively been described as having T = 1 or T = 2 symmetry, although it is important to note these are essentially academic interpretations of a similar particle architecture. In some but not all of these viruses the inner capsid is also surrounded and stabilised by outer protein layers that show T = 13 icosahedral symmetry and therefore a remarkable symmetry mismatch with the inner layer. However, some families of dsRNA viruses do have a different basic structure, suggesting a distinct evolutionary pathway. These include the unencapsidated hypoviruses (Hillman et al., 2000), the rod shaped varicosaviruses (Mayo, 2000) and even the icosahedral birnaviruses (Leong et al., 2000), which although they also have T = 13 symmetry, appear to have only a single capsid layer. Many other parallels are evident in some but not all dsRNA viruses, which at least identify common solutions to similar problems and provide further support for a common if in some cases distant ancestry. For example, the virus core associated RNA polymerase (replicase/transcriptase) of many of these viruses has a structure similar to other polymerases and uses both ss and dsRNA templates. Characteristically members of the family Reoviridae have polymerases with a fully conservative mechanism (see Tables 3–9). While cystoviruses and birnaviruses are semi-conservative. The birnavirus polymerase is also unusual, existing as a VPg covalently bound to the five prime termini of the viral RNA, with no evidence of RNA capping (despite being a eucaryotic virus) (Table 8).

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Table 3 Identification of different (comparable) proteins of the dsRNA viruses Virus (genus: family)

Innermost capsid shell (120 copies per virion) (T1 or T2)a

Polymerase (Cap-Pol, Gag-Pol) VP1 (Pol, VPg)

Capsid protein (Cap, Gag)

P2 (Pol) ␭ 3 (Pol) VP1 (Pol) VP1 (Pol) VP1 (Pol) VP1 (Pol) VP2 (Pol) VP2 (Pol)

P1 (T2) ␭1 (Hel) VP3 (T2) VP2 (T1)

VP1 (T1)

Capping enzyme (CaP)

␭ 2 (CaP) VP4 (CaP) VP3 (CaP) VP5 (CaP) VP1 (CaP) VP3 (CaP)

VP1 (Pol) P1 (Pol) P4A (Pol)

Helicase (Hel)

P4 (Hel) ␭1 (Hel) VP6 (Hel) NSP2 (VIP) VP10 (Hel) VP3 (Hel) VP3 (Hel)

Viral inclusion body (viroplasm) matrix protein (ViP)

Second capsid shell protein (T = 13 symmetry) (T13)

Tubule forming protein (TuP)

P8 (T13) NS2 (ViP) NSP2 (VIP)

VP7 (T13) VP6 (T13)

NS1 (TuP)

ViP P3

P5 (CaP) P5 (CaP)

P8 (T13)

a The 120 protein molecules that form the innermost complete capsid shell of many of the icosahedral viruses, have been alternatively described as being arranged with either T = 1 or T = 2 icosahedral symmetry. It is important to note that these are simply different interpretations and descriptions of essentially similar icosahedral structures.

P. Mertens / Virus Research 101 (2004) 3–13

Lettuce big-vein virus (Varicosavirus: ???) Cryphonectria hypovirus 1 (CHV-1) (Hypovirus: Hypoviridae) Gaeumannomyces graminis virus 019/6-A (GgV-019/6-A) (Partitvirus: Partitiviridae) Saccharomyces cerevisiae virus ScV-L1 (SCV-LA) (Totivirus: Totiviridae) Infectious Pancreatic necrosis virus (IPNV) (Aquabirnavirus: Birnaviridae) Pseudomonas phage Phi 6 (Cystovirus: Cystoviridae) Mammalian reovirus-3 (MRV-3) (Orthoreovirus: Reoviridae) Bluetongue virus (BTV) (Orbivirus: Reoviridae) Simian rotavirus A/SA11 (Rotavirus: Reoviridae) Colorado tick fever virus (CTFV) (Coltivirus: Reoviridae) Banna virus (BAV) (Seadornavirus: Reoviridae) Golden Shiner virus (GSV) (Aquareovirus: Reoviridae) Bombyx mori cypovirus 1 (BmCPV-1) (Cypovirus: Reoviridae) Rice black streaked dwarf virus (RBSDV) (Fijivirus: Reoviridae) Rice dwarf virus (RDV) (Phytoreovirus: Reoviridae) Rice ragged stunt virus (RRSV) (Oryzavirus: Reoviridae)

Polymerase (Pol)

Table 4 The dsRNA genome segments and proteins of Pseudomonas phage Phi 6 (genus Cystovirus: family Cystoviridae) Open reading frames (ORFs)

Proteins (protein structure/functiona )

Size ‘aa’ (kDa)

Protein copy number per particle

Location

Function

L (6374)

Gene 1

P1 (T = 2)

769 (84.986)

120

Inner particle shell

Gene 2

P2 (Pol)

664 (74.791)

12

Inner particle

Gene 4

P4 (NTPase)

331 (35.032)

72

Inner particle at five-fold axes

Gene 7

P7 (assembly factor)

160 (17.169)

60

Inner particle

Gene 14

P14

61 (6.810)

0

Non-structural

Self assembles with proteins P2, P4 and P7 into procapsid particles. Controls procapsid size and organization. Binds minor-proteins and RNA. Conserved in dsRNA viruses. Semi-conservative RNA dependent RNA polymerase using ss and dsRNA templates. Three-dimensional structure homologous to HIV reverse-transcriptase and hepatitis C polymerase. Binding sites for nucleic acid, NTPs, Mn and Mg ions. Hexameric NTPase, symmetry mismatched position. Powers the packaging of ssRNA genomic precursors and is involved in transcription. Nucleates the assembly of the procapsid with P1. Dimeric, stabilizes the procapsid. Increases the assembly rate of the procapsid in vitro. Nonessential.

Gene 3

P3 (spike)

648 (69.178)

Gene 6

P6 (fus)

167 (17.229)

Gene 10 Gene 13

P10 (lys) P13

42 (4.272) 72 (7.649)

Gene 5

P5 (mur)

219 (24.018)

Gene 8

P8 (T = 13)

148 (15.873)

600

Gene 9 Gene 12

P9 (env) P12 (assembly)

89 (9.482) 195 (20.293)

0

M (4063)

S (2948)

Extends from virion surface Membrane Membrane Membrane Between the membrane and NC Second protein layer

Membrane Non-structural

Binds to the receptor, the pilus IV of Pseudomonas syringae. Attached to the integral membrane protein P6. Integral membrane protein that has membrane fusion activity and binds the receptor-binding protein, P3. Needed for cell lysis. Minor, nonessential membrane protein. Muralytic enzyme exposed to host peptidoglycan layer after membrane fusion during viral entry. Also used to lyse the host cell late in infection to release the virus. A trimer that forms the T = 13 nucleocapsid shell around the dsRNA-containing inner particle (core). During entry P8 drives the penetration of the nucleocapsid into the cytoplasm. Major membrane protein, essential for membrane formation Assembly factor active in viral membrane morphogenesis.

P. Mertens / Virus Research 101 (2004) 3–13

DsRNA segment (size, bp)

Table constructed with information provided by Sarah Butcher and Dennis Bamford (Bamford, 2000), available as a webpage with references on: http://www.iah.bbsrc.ac.uk/dsRNA virus proteins/ Cystovirus.htm. a To facilitate comparisons of proteins across species, genera and families, protein structure/function indicators have been added to protein names, e.g. (Pol): RNA polymerase; (T = 2): capsid protein organized with icosahedral T = 2 symmetry; (T = 13): capsid protein organized with icosahedral T = 13 symmetry.

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P. Mertens / Virus Research 101 (2004) 3–13

Table 5 The dsRNA genome segments and proteins of mammalian reovirus-3 (MRV-3) (genus Orthoreovirus: family Reoviridae) dsRNA (size, bp)

ORFs (bp inclusive)

Proteins (protein structure/functiona )

L1 (3854)

19–3819

␭3 (Pol)

1267 (142)

Core

Fully conservative RNA dependent RNA polymerase

L2 (3916)

14–3880

␭2 (CaP)

1289 (144)

60

Core spike

Guanylyltransferase, methyltransferase “turret” protein. Comparable to the cypovirus turret protein VP3 (CaP). Virus species specific antigen.

L3 (3901)

14–3838

␭1 (Hel)

1275 (143)

120

Core

Inner capsid structural protein, binds dsRNA and zinc, putative NTPase, helicase and 5 -triphosphate phospohydrolase.

M1 (2304)

14–2221

␮2

736 (83)

12

Core

NTPase, influences the morphology of inclusion bodies, interacts with cytoskeleton.

M2 (2203)

30–2153

␮1

708 (76)

30

Outer capsid

␮1C (T13)

667 (72)

600

Multimerizes with ␴3. Cleaved to ␮1C and ␮1N, which assume T = 13 symmetry in the outer capsid ␮1C is cleaved to ␦ and ␾ during the entry process. Myristoylated N-terminus, membrane penetration.

␦ ␾ ␮1N

539 (59) 128 (13) 42 (4)

600 0

N/S

N/S

Binds ssRNA and virus cores, primary determinant of inclusion body formation, interacts with ␮2 and ␴NS. Phosphoprotein, coiled coil motifs, transcriptase interaction, genome packaging? ␮NSC is from alternate translation start site, unknown function.

M3 (2241)

19–2181

␮NS

Protein size aa (kDa)

Protein copy number per particle 12

721 (80)

Location

Protein function

␮NSC

681 (75)

0

13–1377

␴1

455 (49)

36

71–430

␴1s

120 (16)

0

N/S

S2 (1331)

19–1272

␴2

418 (47)

150

core

Inner capsid structural protein, weak dsRNA-binding, morphogenesis?

S3 (1189)

28–1125

␴NS

366 (41)

0

N/S

ssRNA-binding, associates with ␮NS during inclusion body formation, genome packaging?

S4 (1196)

33–1127

␴3

365 (41)

600

Outer capsid

dsRNA-binding, multimerizes with ␮1C, nuclear and cytoplasmic localisation, translation control. Virus species specific antigen.

S1 (1416)

Outer capsid

Cell attachment protein, homo-trimer, haemagglutinin, type-specific antigen, possible glycosyl hydolase activity, induces apoptosis. Interacts with neutralising antibodies. Induces cytotoxic T-cell response. Basic protein, nonessential, blocks cell cycle progression. Induces cytotoxic T-cell response.

Table constructed using data supplied by Mertens et al. (2000) with updates by Roy Duncan, Jim Chappell and Terry S. Dermody. Available as a webpage on: http://www.iah.bbsrc.ac.uk/dsRNA virus proteins/Orthoreovirus.htm. a Protein structure/function: RNA polymerase: A(Pol)@; capping enzyme: A(CaP)@; Virus structural protein with T = 13 symmetry: A(T13). Protein with helicase activity: “(Hel)”. Other species within the genus may have proteins with significant differences in sizes.

The outer capsid layers 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. Indeed, the effective dissemination of virus particles within and between individual hosts, attachment and penetration of target cells and even moving such a relatively large structure across the host cell membrane, represent a set of significant challenges for the protein components and organisation of these outer capsid layers, that are unique to each host species (Chandran and Nibert, 2003). The molecules that make up the outer layers of the virus are also responsible for protecting the virus core, keeping it in a stable, if dormant state between the infection of one cell

and the next. However, the processes of virus dissemination and initiation of infection are likely to bring them into contact with host defences, ranging from proteases, to high or low pH, and specific antiviral antibodies, or cellular components of the host’s immune system. As a consequence many of the outer capsid layers and proteins show specific adaptations to their host species and a greater degree of diversity in organisation and structure, than those of the inner core, the transcriptase complex, or non-structural proteins. Although as mentioned already many of the outer capsid layers still show elements of T = 13 icosahedral symmetry (Table 3). In particular, those dsRNA viruses that infect vertebrates, face the hosts immune system and their outer coat proteins

Table 6 The dsRNA segments and proteins of bluetongue virus serotype 10 (BTV-10) (BTV: genus Orbivirus: family Reoviridae) ORFs (bp)

Proteins (function)b

Protein size aa (Da)

Copy number/ particle

Location

Functions and properties

1 (3954)

12–3917

VP1 (Pol)

1302 (149,588)

∼12

Inner surface of sub-core

RNA dependent RNA polymerase.

2 (2926)

20–2887

VP2

956 (111,112)

180

Outermost capsid protein

Outer layer of the outer capsid. Controls virus serotype. Cell attachment protein. Involved in determination of virulence, readily cleaved by proteases. Most variable protein. Reacts with neutralising antibodies. Trimer.

3 (2770)

18–2720

VP3 (T2)

901 (103,304)

120

Sub-core capsid shell

Forms the innermost sub-core capsid shell. T = 2 symmetry, controls overall size and organisation of capsid structure. RNA binding. Interacts with minor internal proteins (VP1, VP4 and VP6).

4 (1981)

8–1940

VP4 (Cap)

644 (76,433)

20

Inside sub-core

Dimer, transmethylase 1 and 2, guanylyltransferase (capping enzyme), nucleotide phosphohydrolase. Forms links to NDPs and NTPs.

5 (1769)

35–1690

NS1 (TuP)

552 (64,445)

0

Tubules

Forms tubules of unknown function in the cell cytoplasm. These are characteristic of orbivirus replication. Interacts with cytoskeleton.

6 (1638)

30–1607

VP5

526 (59,163)

360

Outer capsid

Inner layer of the outer capsid, glycosylated, helps determine virus serotype, variable protein. Trimer. Can mediate cell fusion. Probable role in cell entry.

7 (1156)

18–1064

VP7 (T13)

349 (38,548)

780

Outer core

Trimer, forms outer core surface, which can bind dsRNA. T = 13 symmetry. Not exposed on virion surface. In some species (AHSV) it can form flat hexagonal crystals. Involved in cell entry and high core particle infectivity in insect vector cells. Reacts with “core neutralising” antibodies, Immuno dominant virus-species specific antigen. Binds to glycoaminoglycans.

8 (1124)

20–1090

NS2 (ViP)

357 (40,999)

Viral inclusion body

Viral inclusion body matrix protein, ssRNA binding, phosphorylated. Usually regarded as non-structural but small amounts may be associated with the outer capsid, removed by protease treatment.

9 (1046)

16–1002

VP6 (Hel) VP6a

328 (35,750)

60

Inside sub-core

ssRNA and dsRNA binding, Helicase, NTPase.

10 (822)

20–706 59–706

NS3 NS3a

229 (25,572) 216 (24,020)

0 0

Viral protein in cell membrane

Glycoproteins, membrane proteins, involved in cell exit. Variable protein, May be involved in determination of virulence and vector competence. Cytotoxic, can disrupt cell membranes.

0?

9

Table constructed using data provided by Mertens et al. (2000) with updates. Please mail any additional data to add to this table, or suggested corrections to: [email protected]. Available as a webpage (with references): www.iah.bbsrc.ac.uk/dsRNA virus proteins/Orbivirus.htm. a Based on the genome segment order during agarose gel electrophoresis. Abbreviation: AHSV: African horse sickness virus. b To facilitate identification and comparisons of proteins Protein function is indicated by abbreviations: RNA polymerase: (Pol); capping enzyme (guanylyltransferase) + (CaP); helicase: (Hel); sub-core protein with T = 2 symmetry: (T2); Protein with T = 13 symmetry: (T13); viral inclusion body matrix protein: (ViP); tubule protein: (TuP).

P. Mertens / Virus Research 101 (2004) 3–13

Genome segmenta (size, bp)

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Table 7 The dsRNA segments and proteins of simian rotavirus A/SA11 (genus Rotavirus: family Reoviridae) ORFs (bp)

Proteins (protein functionb )

Protein size aa (Da)

1 (3302)

18–3282

VP1 (Pol)

1088 (125005)

12

2 (2690)

17–2659

VP2 (T1)

880 (102431)

120

3 (2591)

50–2554

VP3 (Cap)

835 (98120)

12

Inner capsid, five-fold axis

4 (2362)

10–2337

VP4

776 (86782)

120

–

VP5∗

Outer capsid (spike)

529, 247–776 (60000)

–

VP8∗

VP4 Dimers form outer capsid spike. Interacts with VP6. Virus infectivity enhanced by trypsin cleavage of VP4 into VP5∗ and VP8∗ .. Hemagglutinin. Cell attachment protein. P-type neutralization antigen. VP5∗ permeabilizes membranes. Crystal structure of VP8 fragment (galectin fold). TRAF2 signaling. Protection.

247, 1–247 (28000)

5 (1611)

31–1515

NSP1

495 (58654)

N/S

6 (1356)

24–1214

VP6 (T13)

397 (4816)

7 (1105)

26–970

NSP3

315 (34600)

0

N/S

8 (1059)

47–997

NSP2 (VIP)

317 (36700)

0

N/S

9 (1062)

49–1026

VP7

326 (7368)

10 (751)

41–569

NSP4

175 (20290)

0

N/S

Associates with cytoskeleton. Extensive sequence diversity between strains. Two conserved cysteine-rich zinc-finger motifs. Virus specific 5 -mRNA binding. Interacts with host IFN regulatory factor 3. Major virion protein. Middle capsid structural protein. Homotrimeric 4◦ structure. Subgroup antigen. Myristoylated. Protection (? Mechanism). Crystal structure available. Hydrophobic. Homodimer. Virus-specific 3 -mRNA binding. Binds eIF4G1 and circularizes mRNA on initiation complex. Involved in translational regulation and host shut-off. Crystal structures: NSP3 NH3 fragment with 3 -viral RNA and NSP3 COOH fragment with eIF4G fragment. Non-specific ssRNA-binding. Accumulates in viroplasm. Involved in viroplasm formation with NSP5. NTPase activity. Helix destabilization activity. Functional octamer. Binds NSP5 and VP1. Regulates NSP5 autophosphorylation. Crystal structure (HIT-like fold). Outer capsid structural glycoprotein. G-type neutralization antigen. N-linked high mannose glycosylation and trimming. RER transmembrane protein, cleaved signal sequence. Ca2+ binding. Protection. Enterotoxin. Receptor for budding of double-layer particle through ER membrane. RER transmembrane glycoprotein. Ca2+ /Sr2+ binding site. N-linked high mannose glycosylation. Protection. Host cell (Ca2+ ) mobilization.

11 (667)

22–615

NSP5

198 (21725)

0

N/S

80–355

NSP6

92 (11012)

0

N/S

Copy number/ particle

0

780

780

Location

Protein functions and properties

Inner capsid, five-fold axis Inner capsid

RNA-dependent RNA polymerase. Part of minimal replication complex. Virus specific 3 -mRNA binding. Part of virion transcription complex with VP3. Inner capsid structural protein. Non-specific ss & dsRNA-binding activity. Myristoylated. Cleaved. Part of minimal replication complex. Leucine zipper. Interacts with VP5. Guanylyltransferase. Methyltransferase. Basic Protein. Part of virion transcription complex with VP1. Non-specific ssRNA binding.

Middle capsid

Outer capsid glycoprotein

Interacts with VP2, NSP2 and NSP6. Homomultimerizes. O-linked glycosylation. (Hyper-) Phosphorylated. Autocatalytic kinase activity enhanced by NSP2 interaction. Non-specific ssRNA binding. Product of second, out-of-frame ORF. Interacts with NSP5. Localizes to viroplasm.

Table constructed using data provided by Mertens et al. (2000). Available with references at www.iah.bbsrc.ac.uk/dsRNA virus proteins/Rotavirus.htm). Please make suggestions for changes or updates to this table by e-mail to Peter Mertens [email protected]. a Segments numbered based on migration of SA11 genome segments in SDS-PAGE gel. Migration order may differ among other members of the genus. b Protein structure/function: RNA polymerase: (Pol); capping enzyme: (CaP); Inner virus structural protein with T = 13 symmetry: (T13); viral inclusion body or viroplasm matrix protein: (ViP). Other species within the genus may have proteins with significant differences in sizes.

P. Mertens / Virus Research 101 (2004) 3–13

Genome segmenta (size, bp)

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Table 8 The dsRNA genome segments and proteins of infectious pancreatic necrosis virus (IPNV) strain Jasper (genus Aquabirnavirus: family Birnaviridae) DsRNA segment (size, bp)

Open reading frames (ORFs)

Proteins (protein Protein Size structure/functiona ) ‘aa’ (kDa)

protein copy number per particle

Location

Protein function and properties

Segment A (3092)

120–3035

Polyprotein

972 (106)

Unknown

Cytoplasm

Pre-VP2

491 (62)

Unknown

Virion

VP2 NS

(54) 221

Unknown 0

Virion Non-structural

VPS

235 (30) 148 (17)

Unknown 0

Internal capsid

Polyprotein precursor of viral proteins VP2, NS and VP3. The exact sites of cleavage of the polyprotein are unknown. Pre-VP2 is cleaved to produce VP2, both forms are found in the virion. VP2 is a major capsid protein, type specific and neutralising antigen. O-linked glysosylation. Apparently unique protease, active site at carboxy end, NS processed to NSt and Nsta. Group specific (cross reactive) antigen. Positively charged non-structural protein from separate open reading frame.

VP1 (Pol, VPg)

845 (94)

Unknown

Virion both as a free protein and genome linked form

68–511 Segment B (2784)

101–2638

RNA dependent RNA polymerase, unlike those of the members of the Reoviridae, it has a semi-conservative mechanism. Covalently linked via a ‘G’ residue (VPg), can self-guanylate, but there is no evidence for guanylyltransferase activity, or capping.

Available as a webpage with updates on: www.iah.bbsrc.ac.uk/dsRNA virus proteins/Aquabirnavirus-IPNV.htm, If you have suggestions for additions or corrections to this page, please mail them to [email protected]. a To facilitate comparisons of proteins across species, genera and families, protein structure/function indicators have been added to protein names, e.g. (Pol): RNA polymerase; genome linked protein: (VPg) (Leong et al., 2000). Table 9 The dsRNA segments and proteins of Saccharomyces cerevisiae virus ScV-L1 (SCV-LA) (genus Totivirus: family Totiviridae) Single genome segment (size, bp)

ORFs (bp) (inclusive)

Proteins (protein function)

Protein size aa (kDa)

4579

30–2072

Capsid protein (Cap, Gag)

680 (76.1)

1939–4579

Polymerase (Cap-Pol, Gag-Pol)

1505 (170.5)

Copy number/particle

Location

Functions and properties

120

Virion

One or two molecules

Virion

Major capsid polypeptide. May be phosphorylated. RNA dependent RNA polymerase (replicase transcriptase) (fully conservative), RNA binding. Packaging of viral plus strands.

These figures are for ScV-L1 (ScV-LA), although there are similar proteins known for other totiviruses. The stochiometery was determined by cryo-electron microscopy and SDS page analysis of capsid proteins. Table based on information from the Abstracts for the 7th International Symposium for dsRNA viruses Aruba 2000 (Wickner et al., 2000). Updates available at www.iah.bbsrc.ac.uk/dsRNA virus proteins/saccharomyces-cerevisiae-virus.htm. Please make suggestions for changes or updates to this table by e-mail to [email protected].

are subject to antibody selective pressure, which may by itself lead to higher levels of amino acid sequence diversity (as observed in the outer capsid proteins VP2 and VP5 of the orbiviruses (Table 6), and VP4 and VP7 of rotaviruses (Table 7)).

4. Expression strategies for viral proteins The genomes of the icosahedral dsRNA viruses have a size that is limited by the volume that can be effectively enclosed

by the inner capsid shell, while still allowing active transcription of the viral genome. Many of the dsRNA viruses have segmented genomes (Tables 1 and 2). This provides a mechanism that conveniently breaks up the total translation products from the genome into several distinct proteins. Individual genome segments can also be exchanged when compatible viruses (usually closely related) infect the same cell, effectively creating a mechanism that not only increases genetic diversity in the virus population, but potentially also reduces the effect of lethal mutations to single genes, rather than the whole virus genome.

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P. Mertens / Virus Research 101 (2004) 3–13

Each segment of the genome is transcribed separately to make full-length +ve sense RNA copies. These viral mRNAs function not only as translation templates, but also as templates for full-length −ve strand synthesis, during formation of the progeny virus genome. This replication mechanism together with the highly processive nature of the RNA polymerases (Laurila et al., 2002), may help explain why translationally functional mRNAs, that are incomplete copies of whole genome segments, have only been found with a single virus (leishmaniavirus) (Patterson et al., 2003). Different mRNAs are generated in different molar amounts, depending at least partially on their size (more copies of smaller segments), providing considerable differential control over the relative expression levels from individual viral genes. In the Reoviridae these differences in transcription frequency result from the association of each genome segment with only a single recycling replicase complex, within the transcriptionally active compartment of the virion. However, there can only be a maximum of 12 of these complexes (situated at the 12 internal vertices of the icosahedron), which also restricts the maximum number of genome segments to 12 and could therefore impose some limitation on the maximum number of viral proteins. Indeed most of the genome segments of the members of the family Reoviridae represent single genes, with a single long ORF and relatively short non-coding regions. In these cases the protein produced may be a straightforward translation product of the mRNA. The mRNAs of some genome segments are translated much more efficiently, providing a significant further level of differential control of individual protein expression that is largely independent of genome segment size (Mitzel et al., 2003). However, possibly because of the limitations imposed by genome segment number, many of the dsRNA viruses (particularly those with only 1, 2 or 3 genome segments) have evolved mechanisms that generate multiple different proteins from single mRNA species. These mechanisms include: sequential and unrelated ORFs on a single mRNA (e.g. genome segment 7 of rice black streaked dwarf virus (a Fijivirus), and all three genome segments of phi 6 (Table 4)); Post translational modification of a polyprotein (as seen in the birnavirus genome segment 1 (Table 8)); Translation ‘skip’ sequences that release a truncated amino terminal translation product, even though the ribosome continues to make the polypeptide chain (e.g. cypovirus-1 segment 5); Multiple in frame initiation sites generating related proteins of different sizes (e.g. orbivirus segment 10 (Table 6)); Alternative reading frames (Orthoreovirus segment S1, rotavirus segment 11). There is even a possibility of an ORF on the −ve RNA strand of some cypovirus genome segments, although our current model for virus replication does not allow for release of individual −ve sense RNA strands. If a translation product from this −ve strand ORF can be detected in infected cells, this may therefore require some revision of the existing replication model.

5. Web based information With biochemical and sequence data concerning viral proteins/RNAs becoming available ever more rapidly, it is useful to be able to quickly identify and compare the equivalent proteins and genes from different viruses. However, different RNA/protein nomenclatures have arisen, not only for the different genera of dsRNA viruses, but frequently even within a single virus genus or species. Therefore, in order to facilitate comparisons and provide information concerning cognate viral proteins of the dsRNA viruses, a number of webpages have been established that are accessible via the dsRNA virus website at: www.iah.bbsrc.ac.uk/dsRNA virus proteins/. This displays a series of tables that were originally developed for the 7th International dsRNA Virus Symposium held on Aruba, 2000, containing data concerning representative members of the Totiviridae, Birnaviridae, Cystoviridae and each of the 10 different genera of the Reoviridae. Each table shows the current protein nomenclature, together with indicators of functional roles, where known. These web tables include links to many of the more recent relevant publications as well as to other sources of information. Additional tables and links will be included, as more information becomes available (for example, tables describing the RNAs and proteins of the newly proposed genera of insect and fungal reoviruses as well as the other families of dsRNA viruses, are currently under construction). Each page is also available as a Word file, intended to provide a readily accessible source of information that can be regularly updated. The tables that are included here as examples illustrate the information currently provided for: Pseudomonas phage Phi 6 (Table 4); mammalian reovirus (Table 5); bluetongue virus (Table 6); rotavirus (Table 7); infectious pancreatic necrosis virus (Table 8) and Saccharomyces cerevisiae virus LA (Table 9).

References Bamford, D.H., 2000. Family Cystoviridae. In: Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Calisher, C.H., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, pp. 389–393. Chandran, K., Nibert, M.L., 2003. Animal cell invasion by large nonenveloped viruses: reovirus delivers the goods. Trends Microbiol. 11, 374–382. Gitlin, L., Andino, R., 2003. Nucleic acid-based immune system: the antiviral potential of mammalian RNA silencing. J. Virol. 77, 7159– 7165. Ghabrial, S.A., Bozarth, R.F., Buck, K.W., Martelli, G.P., Milne, R.G., 2000. Family Partitiviridae. In: Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Calisher, C.H., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, pp. 503–513.

P. Mertens / Virus Research 101 (2004) 3–13 Ghabrial, S.A., Castón, J., Jiang, D., Carracoa, J., 2003. Molecular Characterisation and particle structure of Penicillium chrysogenum virus: a dsRNA mycovirus with multipartite genome. Final Programme and Abstracts, 8th International Symposium on Double-stranded RNA Viruses (Institution Superiore di Sanità) (Abstract W1.1 pp1). Goldbach, R., Bucher, E., Prins, M., 2003. Resistance mechanisms to plant viruses: an overview. Virus Res. 92, 207–212. Hillman, B.I., Fulbright, D.W., Nuss, D.L., van Alfen, N.K., 2000. Family Hypoviridae. In: Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Calisher, C.H., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, pp. 515–520. Jacobs, B.L., Langland, J.O., 1996. When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology 219, 339–349. Laurila, M.R., Makeyev, E.V., Bamford, D.H., 2002. Bacteriophage phi 6 RNA-dependent RNA polymerase: molecular details of initiating nucleic acid synthesis without primer. J. Biol. Chem. 277, 17117– 17124. Leong, J.C., Brown, D., Dobos, P., Kibenge, F.S.B., Ludert, J.E., Muller, H., Mundt, E., Nicholson, B., 2000. Family Birnaviridae. In: Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Calisher, C.H., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, pp. 481–490. Mayo, M.A., 2000. Genus Varicosavirus. In: Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Calisher, C.H., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B., Virus Taxonomy. Seventh Report of the Inter-

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national Committee for the Taxonomy of Viruses. Academic Press, pp. 521–523. Mertens, P.P.C., Arella, M., Attoui, H., Belloncik, S., Bergoin, M., Boccardo, G., Booth, T.F., Chiu, W., Diprose, J.M., Duncan, R., Estes, M.K., Gorziglia, M., Gouet, P., Gould, A.R., Grimes, J.M., Hewat, E., Hill, C., Holmes, I.H., Hoshino, Y., Joklik, W.K., Knowles, N., López Ferber, M.L., Malby, R., Marzachi, C., McCrae, M.A., Milne, R.G., Nibert, M., Nunn, M., Omura, T., Prasad, B.V.V., Pritchard, I., Samal, S.K., Schoehn, G., Shikata, E., Stoltz, D.B., Stuart, D.I., Suzuki, N., Upadhyaya, N., Uyeda, I., Waterhouse, P., Williams, C.F., Winton, J.R., Zhou, H.Z., 2000. Family Reoviridae. In: Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Calisher, C.H., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, pp. 395–480. Mertens, P.P.C., Mellor, P.S., 2003. Bluetongue State Vet. J. 13, 18–25. Mitzel, D.N., Weisend, C.M., White, M.W., Hardy, M.E., 2003. Translation regulation of Rotavirus gene expression. J. Gen. Virol. 84, 383–391. Patterson, J., Cairion, R., Ro, Y., Ticer, A., 2003. Biochemical analysis of Leishmaniavirus specific proteolysis and Leishmaniavirus specific endoribonuclease activity. In: 8th International Symposium on Double -Stranded RNA Viruses, Final Programme and Abstracts. Instituto Superiore di Sanita, Rome, p. 51. Wickner, R.B., Ghabrial, S.A., Bruenn, J.A., Buck, K., Patterson, J.L., Stuart, K.D., Wang, C.C., 2000. Family Totiviridae. In: Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Calisher, C.H., Carsten, E.B., Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, pp. 491–501.