Closteroviridae

Giovanni P Martelli, Dipartimento di Protezione delle Piante, University of Bari, Italy ge tale, INRA, Villenave dOrnon, France Thierry Candresse, Station de Pathologie Ve
The Closteroviridae is a family of plant viruses with very large, positive-sense, singlestranded genomes. It comprises at least two genera, Closterovirus and Crinivirus, members of which are major insect-transmitted pathogens, causing extensive damage to sugarbeet, citrus, grape and vegetable crops.

Secondary article
Article Contents
. Historical Perspective . Classification . Structure of Virus Particles . Structure of the Virus Genome . Pathogenesis . Transmission and Epidemiology . Control

Historical Perspective
In 1976, the International Committee on Taxonomy of Viruses (ICTV) recognized a taxonomic group of plant viruses with exuous, helically constructed lamentous particles with open structure and conspicuous cross banding and gave it the name of Closterovirus (from kloster, Greek for thread). Initially, the Closterovirus Group contained 13 members that were subdivided into two subgroups, based on aphid transmissibility and size of the capsid protein. In 1982, the group was further subdivided into three, in recognition of the wide dierences in particle length that ranged from 720 to 2000 nm. By 1988, there were 21 closteroviruses although four previous members with particles 600800 nm long were reclassied in a new Group called Capillovirus. In 1993, there were 31 recognized closteroviruses, subdivided into three subgroups. However, shortly afterwards, the ICTV extended the familygenusspecies classication system to plant viruses, so that the former Closterovirus Group became the genus Closterovirus (Candresse and Martelli, 1995). Meanwhile, more detailed molecular information on genome structure and composition of a number of members of the genus, and knowledge on their epidemiology and relationships with vectors, led to a critical reappraisal of closterovirus classication. Thus, two new independent and separate genera (Trichovirus and Vitivirus) were established following further splitting, and the genus Closterovirus was raised to the rank of family (Closteroviridae), comprising two genera with monopartite (Closterovirus) and bipartite (Crinivirus from crinis, Latin for hair) genome (Martelli, 1997). 2. Very large, positive-sense, single-stranded RNA genome with an organization (number and order of genes) distinct from those of other plant viruses. 3. Possession of unique genes coding for a homologue of the cellular HSP70 heat shock protein and for a duplicated, diverged copy of the capsid protein. 4. Possession of a conserved ve-gene module between the genes coding for the HSP70 protein analogue and the capsid protein. 5. Close phylogenetic relationships in replication-related proteins (methyltransferase, helicase and RNA-dependent RNA polymerase). 6. Genome expression strategy based on ribosomal frameshift, proteolytic processing and the production of subgenomic messenger RNAs. 7. Induction of specic cytopathic structures consisting of cytoplasmic aggregates of virus particles intermingled with single or clustered membranous vesicles. 8. Specic tissue tropism (members are mostly phloemlimited). 9. Natural transmission by aphids, mealybugs or whiteies in a semi-persistent manner; experimental transmission by mechanical inoculation very dicult or not possible.

The genera
The family Closteroviridae is divided into at least two genera on the basis of genome size and organization, biological properties (type of vector, host range), and the results of phylogenetic analyses. The genera are: 1. Genus Closterovirus, typied by Beet yellows virus (BYV), comprising species with particle length above 1200 nm, monopartite genome 15.519.3 kb in size, transmitted by aphids, pseudococcid and coccid mealybugs, or whiteies. 2. Genus Crinivirus, typied by Lettuce infectious yellows virus (LIYV), comprising species with particles with two modal lengths (650800 and 700900 nm),
1

Classification
Defining characteristics
Traits that characterize the family and that are the basis of the current classication are: 1. Morphology of virus particles.

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bipartite genomes 1517 kb (78.2 1 88.7 kb) in size, transmitted by whiteies. The members of the genera Closterovirus and Crinivirus are listed in Table 1.

Structure of Virus Particles

Virions are helically constructed with a pitch of the primary helix in the range of 3.43.8 nm and about 10 protein subunits per turn (Tollin and Wilson, 1988). The very exuous and open structure of the particles is the most conspicuous trait of members of the family Closteroviridae, notwithstanding the morphological resemblance to exuous lamentous particles of other plant viruses in the genera Trichovirus, Capillovirus, Vitivirus and Foveavirus. Virus particles have a diameter of about 12 nm and various lengths. The fragility of virions and a tendency to end-to-end aggregation, mean that often a range of lengths is given for single viruses. However, smaller than fulllength particles exist in nature (e.g. of CTV), and probably
Table 1 Members of the family Closteroviridae Denitive species Genus Closterovirus Aphid-transmitted

encapsidate subgenomic messenger RNAs or defective interfering RNAs. The capsid proteins (CPs) of most members of the family are 2230 kDa in size. Notable exceptions are the CPs of the mealybug-transmitted viruses LChV, GLRaV-1 and GLRaV-3 and of other grapevine leafroll-associated viruses, that have molecular masses of up to 46 kDa. In all viruses sequenced so far, two types of CPs were identied: the genuine CP and a CP analogue that probably arose by gene duplication followed by sequence divergence (Boyko et al., 1992). With BYV and CTV, the duplicate CP coats an extremity of the virions, forming a distinct structure for which the terms rattlesnake or heterodimeric were coined. Given the consistent presence of CP analogues in all sequenced viruses, this structure probably is a general characteristic of the family (Agranovsky et al., 1995).

Structure of the Virus Genome

Members of the family Closteroviridae have the largest genomes among plant viruses (15.319.3 kb), probably

Tentative species

Beet yellows virus (BYV), Beet yellow stunt virus Clover yellows virus (CYV), Dendrobium vein (BYSV), Burdock yellows virus (BuYV), Carnanecrosis virus (DVNV), Heracleum virus 6 tion necrotic eck virus (CNFV), Carrot yellow (HV6) leaf virus (CYLV), Citrus tristeza virus (CTV), Wheat yellow leaf virus (WYLV) Mealybug-transmitted Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine leafroll associated virus 1 (GLRaVLittle cherry virus (LChV) 1), Pineapple mealybug wilt-associated virus 1 (PMWaV-1), Pineapple mealybug wiltassociated virus 2 (PMWaV-2), Sugarcane mild mosaic virus (SMMV) Whitey-transmitted Beet pseudoyellows virus (BPYV) Diodea vein chlorosis virus (DVCV), Cucurbit chlorotic spot virus (CCSV) Vector unknown Grapevine leafroll-associated virus 2 (GLRaV-2) Alligator weed stunting virus (AWSV), Festuca necrosis virus (FNV), Grapevine leafroll associated virus 4 (GLRaV-4), Grapevine leafroll associated virus 5 (GLRaV-5), Grapevine leafroll associated virus 6 (GLRaV-6), Grapevine leafroll associated virus 7 (GLRaV-7), Megakepasma mosaic virus (MeMV) Genus Crinivirus Abutilon yellows virus (AYV), Cucurbit yellow stunting disorder virus (CYSDV), Lettuce chlorosis virus (LCV), Lettuce infectious yellows virus (LIYV), Tomato chlorosis virus (ToCV), Tomato infectious chlorosis virus (TICV), Sweet potato chorotic spot virus (SPCSV)

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because of sequence duplication and the acquisition of nonviral coding sequences (protease, HSP70 protein) via RNA recombination. This may also explain the dierences in genome organization between genera and members of the same genus. Both monopartite and bipartite genomes consist of positive-sense, single-stranded RNA that accounts for about 56% of the particle weight. The genome is probably capped at the 5 end, is not polyadenylated and has no transfer RNA-like structure at the 3 end, but may contain several hairpin structures. Genomic RNAs of BYV, CTV and LIYV are infectious, and have messenger activity in vitro. To date, complete or nearly complete genomic sequences have been determined for BYV, LIYV, LChV, GLRaV-2, GLRaV-3 and CTV (three isolates from Florida, Israel and California). The 3 terminal sequence is available for the closteroviruses BYSV, CNFV, BPYV, GLRaV-1 and the criniviruses TICV and SPCSV. The genomes of both genera are characterized by the presence of unique genes coding for a homologue of the HSP70 proteins and for an analogue of the coat protein. The HSP70-related protein gene, rst reported for BYV (Agranovsky et al., 1991), has now been identied in the closteroviruses CTV, LChV, BPYV, BYSV, CCSV, CNFV, GLRaV-1, GLRaV-2, GLRaV-3, GLRaV-4, GLRaV-5, GLRaV-7, PMWaV-1 and PMWaV-2 and the criniviruses CYSDV, TICV, LIYV, LCV and ToCV. The functions postulated for this protein are: mediation of cell-to-cell movement through plasmodesmata, involvement in the assembly of multisubunit complexes for genome replication and/or subgenomic RNAs synthesis, or assembly of viruses particles. Recent experimental evidence with BYV mutants demonstrated the role of the HSP70 analogue in cell-to-cell movement of the virus. The duplication of the capsid protein gene seems to be the only example of such condition among viruses with elongated particles. The capsid proteins of BYV and CTV, and their homologues, show a signicant degree of sequence conservation and the duplicate copies probably retain the general spatial folding and some crucial properties of the CPs. Interestingly, in aphid-transmitted members of the family (BYV, CTV, BYSV) the CP duplicate is located upstream of the CP gene, whereas the reverse is true with whitey-transmitted (LIYV, SPCSV, CCSV) and mealybug-transmitted (GLRaV-3, LChV) viruses. The genome organizations, i.e. the number and relative position of the ORFs, dier between the genera and/or individual viral species. However, the complex ORF1a ORF1b invariably encodes the replication-related proteins (methyltransferase, helicase and RNA-dependent RNA polymerase, RdRp). Downstream ORFs that encode in 5A to 3 order a 6-kDa small hydrophic protein, the HSP70 homologue, a 5564-kDa product, the CP, and its analogue, form a ve-gene module. This is conserved, with

few modications, among all members of the family analysed so far. Sequenced species of the genus Closterovirus show three types of genome organization exemplied by BYV (Figure 1), CTV and BYSV: (1) The BYV genome contains nine ORFs anked by 5 and 3 untranslated regions (UTR) of 107 and 181 nucleotides, respectively (Agranovsky et al., 1994). (2) CTV has 12 ORFs and UTRs of 107 nucleotides at the 5 end and 275 nucleotides at the 3 end (Karasev et al., 1995). It diers from the BYV genome in having two papain-like protease domains in ORF1a, an extra 5 proximal ORF (ORF2) encoding a 33-kDa product with no similarity to any other protein in databases, and two extra 3 proximal ORFs (ORF9 and ORF11). (3) The BYSV genome has 10 ORFs and a 3 UTR of 241 nucleotides, a length intermediate between that of the BYSV and CTV UTRs (Karasev et al., 1996). A further dierence with the BYV genome rests in the presence of an extra ORF (ORF2) encoding a 30-kDa polypeptide with no similarity to any other protein in the databases. This ORF is located downstream of ORF1b, i.e. in the same position as the unrelated CTV ORF2. Thus, the organization of BYSV genomes is intermediate between that of BYV and CTV, suggesting that these three viruses might represent three distinct stages in closterovirus evolution. The genome of criniviruses (LIYV) is divided between two linear, positive-sense, single-stranded RNAs totalling 15.316 kb in size (Figure 2). Both molecules are needed for infectivity and are separately encapsidated. RNA1 contains three open reading frames, i.e. the typical ORF1a ORF1b complex plus a 3-most ORF coding for a 32-kDa protein with no similarity to any protein in databases. This ORF is similar in size and location to ORF2 of CTV and BYSV but the respective expression products are not related. RNA1 has 5 and 3 UTRs of 97 and 219 nucleotides, respectively (Klaassen et al., 1995). As with other members of the family, the ORF1aORF1b complex codes for the replication-related proteins including the RNA-dependent RNA polymerase (RdRp). RNA2 has seven ORFs anked by a 5 UTR of 326 nucleotides and a 3 UTR of 187 nucleotides. RNA2 contains the ve-gene module which, however, diers from that of members of the genus Closterovirus because of the insertion of an extra

P-Pro

MT

HEL

6 kb

64 kb

CP

21 kb

POL

HSP70

CPd

20 kb

Figure 1 Genome organization of beet yellows virus, the type species of the genus Closterovirus showing the relative position of the ORFs and their expression products. P-Pro, papain-like protease; MT, methyltransferase; HEL, helicase; POL, RNA polymerase; HSP70, heat shock-related protein; CP, capsid protein; CPd, capsid protein analogue. The five boxes with the same colouring represent the five gene block conserved among closteroviruses.

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P-Pro

MT

HEL

31 kb

POL RNA1

5kb

59 kb

CP

26 kb

HSP70

9 kb RNA2

CPd

Figure 2 Genome organization of lettuce infectious yellows virus, the type species of the genus Crinivirus showing the relative position of the ORFs and their expression products. P-Pro, papain-like protease; MT, methyltransferase; HEL, helicase; POL, RNA polymerase; HSP70, heat shock-related protein; CP, capsid protein; CPd, capsid protein analogue.

gene (ORF4) upstream of the CP gene. In all members of the Crinivirus genus the order of the CP and CP duplicate ORFs is reversed compared to that of the aphidtransmitted species of the genus Closterovirus. SPCSV and ToCV have particularly large ORFs for the CP duplicate (7579 kDa), unlike LIYV (52 kDa). The genome expression strategy of all members of the family is based on: (1) proteolytic processing of the polyprotein encoded by ORF1a; (2) +1 ribosomal frameshift for the expression of the RdRp domain encoded by ORF1b; (3) expression of the downstream ORFs via the formation of a nested set of 3 coterminal subgenomic messenger RNAs. This strategy of replication closely resembles that of members of the order Nidovirales (Coronaviridae, Arteriviridae). However, the RNA-dependent RNA polymerases of these viruses belong to the picorna-like supergroup of polymerases, unlike those of closteroviruses, which belong to the alpha-like supergroup.

irregular bundles intermingled with single or clustered membranous vesicles containing a network of ne brils. As these brils resemble double-stranded RNA, the vesicular structures have been proposed to have a role in virus replication (Martelli and Russo, 1984). Phloem localization of closteroviruses, and the damage caused to these tissues, accounts for the symptoms of the yellowing type that are usually shown by infected hosts. The family Closteroviridae comprises members known to infect only weeds, as well as members that have a signicant detrimental eect on crops. Among these, BYV, CTV, CNFV, the whitey-transmitted viruses at large, and the mealybug-transmitted GLRaV-1 and GLRaV-3 are worth mentioning. Losses of root yield in sugarbeet induced by BYV can be as high as 30%, depending on the time of infection and susceptibility of the aected cultivar. CTV is one of the major citrus pathogens and the cause of a destructive disease known as tristeza. It has caused the death of over 80 million trees worldwide and is still destructive in many countries. Leafroll, one of the economically relevant diseases of grapevines, is elicited at least by GLRaV-1, 2, -3 and -7. Infection induces yield losses of 1070%, lowers the sugar and phenolic compounds content of the berries, reduces graft take and compatibility, and rooting ability. Whitey-transmitted criniviruses are the agents of emerging diseases that cause major losses to a number of vegetable (lettuce, cucurbits, tomato) and industrial (sugarbeet) crops. Severe LIYV infections were held responsible for losses of up to 80% to lettuce in certain years. Epidemic crinivirus outbreaks have been estimated to induce, in a single season, losses of US$ 8 million to lettuce, sugarbeet, melon and squash.

Transmission and Epidemiology

Natural vectors of closteroviruses are aphids, mealybugs and whiteies, all of which transmit in a semi-persistent manner, i.e. long acquisition and transmission periods with a minimum feeding time in the range of 15 and 30 min, respectively, relatively long retention of infectivity (up to 23 days), absence of a latent period, lack of retention through moults, and of transmission to the vector progeny (Martelli, 1997). The molecular bases of this mode of transmission are currently not understood. In particular, the localization of virions in viruliferous vectors is not known, nor are the reasons for the long retention period. The close association of viruses with phloem tissues accounts for some of the transmission traits, such as the length of the acquisition and transmission feeding periods, and the lack of transmission during brief probings. According to a recent hypothesis that needs verication, the CP analogue could facilitate the interaction between virions and the insects foregut.

Pathogenesis
Closteroviruses are primarily localized in the phloem, which can be damaged to varying extents. Plant cells react to infection with both nonspecic modications, such as membrane proliferation, peripheral vesiculation and degeneration of chloroplasts and mitochondria, accumulation of osmiophilic granules, and the formation of family-specic inclusion bodies. These are made up of aggregates of virions and membranous vesicles, or of combinations of the two, in sieve tubes, companion cells and phloem parenchyma, and occasionally the mesophyll and epidermis near local lesions. Viral aggregates can be large enough to occupy the whole cell lumen but, more often, virions occur in loose paracrystalline aggregates or
4

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The range of vectors vary from rather wide to restricted, depending on the virus. Among species of the genus Closterovirus, BYV was shown to be transmitted by 23 aphid species (Myzus persicae and Aphis fabae being the main natural vectors), CTV by seven species (Toxoptera citricida and Aphis gossypii being the most ecient vectors) and a number of other viruses (e.g. CNFV and WYLV) by a single aphid species. Pseudococcid (Planococcus, Pseudococcus, Phenacoccus, Saccharicoccus and Dysmicoccus) and coccid (Pulvinaria, Neopulvinaria and Parthenolecanium) mealybugs were reported to transmit ve members aecting grapevines, sugarcane and pineapple. Transmission is semi-persistent, and may not be vector-specic. For instance, GLRaV-3 can be transmitted by both pseudococcid (Planococcus and Pseudococcus) and coccid (Pulvinaria, Neopulvinaria and Parthenolecanium) mealybugs. Some of the species of the genus Closterovirus and all of those in the genus Crinivirus are transmitted semipersistently by whiteies of the genera Trialeurodes and Bemisia. Persistence and specicity of transmission of whitey-transmitted viruses in their respective vectors have been used as characters for their dierentiation. Long-distance dispersal of closteroviruses is determined by the distribution of contaminated propagating material through trade and other human activities rather than by vector activity.

References
Agranovsky AA, Boyko VP, Karasev AV, Koonin EV and Dolja VV (1991) Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock protein. Journal of Molecular Biology 217: 603610. Agranovsky AA, Koonin EV, Boyko VP et al. (1994) Beet yellows closterovirus: complete genome structure and identication of a leader papain-like thiol protease. Virology 195: 311324. Agranovsky AA, Lesemann DE, Maiss E, Hull R and Atabekov JG (1995) Rattlesnake structure of a lamentous plant RNA virus built of two capsid proteins. Proceedings of the National Academy of Sciences of the USA 92: 24702473. Boyko VP, Karasev AV, Agranovski AA, Koonin EV and Dolja VV (1992) Coat protein gene duplication in a lamentous RNA virus of plants. Proceedings of the National Academy of Sciences of the USA 89: 91569160. Candresse T and Martelli GP (1995) Genus Closterovirus. In: Murphy FA, Fauquet CM, Bishop DHL et al. (eds), Virus Taxonomy. 6th Report of the International Committee on the Taxonomy of Viruses, pp. 461 464. Vienna: Springer-Verlag. Karasev AV, Boyko VP, Nikolaeva OV et al. (1995) Complete sequence of the citrus tristeza virus RNA genome. Virology 208: 511520. Karasev AV, Nikolaeva OV, Mushegian AR, Lee RF and Dawson WO (1996) Organization of the 3 terminal half of beet yellow stunt virus genome and implications for the evolution of closteroviruses. Virology 221: 199207. Klaassen VA, Boeshore ML, Koonin EV, Tian T and Falk BW (1995) Genome structure and phylogenetic analysis of lettuce infectious yellows virus, a whitey-transmitted, bipartite closterovirus. Virology 208: 99110. Martelli GP (1997) Plant virus taxa: properties and epidemiological characteristics. Journal of Plant Pathology 79: 151171. Martelli GP and Russo M (1984) Use of thin sectioning for visualization and identication of plant viruses. Methods in Virology 8: 143224. Tollin P and Wilson HR (1988) Particle structure. In: Milne RG (ed.), The Plant Viruses, vol. 4: pp. 5183. New York: Plenum Press.

Control
In regions where a particular virus is not present, quarantine is the protective measure of choice. In contaminated areas, infections can be contained by controlling insect vector populations and by destruction of virus reservoirs (e.g. beet-free periods for BYV control). Additional measures include sanitary selection and implementation of certication schemes encompassing the production and use of virus-free propagative material. Closterovirus-free plants of several crops have been obtained through meristem tip culture, thermotherapy or a combination of both. When possible, these measures are to be coupled with the use of resistant or tolerant varieties or rootstocks. A search for natural resistance was successful for a few closteroviruses and is currently underway for criniviruses. Cross-protection, i.e. a strategy based on the preinoculation of hosts with mild virus isolates to protect them from the expression of symptoms caused by more severe superinfecting strains, can also be used, as in the case of CTV-induced tristeza disease. Although not without risk, large-scale cross-protection has been successfully used commercially in a number of countries to protect millions of citrus trees from aggressive CTV strains.

Further Reading
Agranosky AA (1996) Principles of molecular organization, expression, and evolution of closteroviruses: over the barriers. Advances in Virus Research 47: 119158. Bar-Joseph M and Murant AF (1982) Closterovirus group. CMI/AAB Description of Plant Viruses, No. 260. Bar-Joseph M, Garnsey SM and Gonsalves D (1979) The closteroviruses: a distinct group of elongated plant viruses. Advances in Virus Research 25: 93168. Bar-Joseph M, Lee RF and Pappu HR (1994) The closteroviruses. In: Singh RP, Singh US and Kohmoto K (eds), Pathogenesis and Host Specicity in Plant Diseases, vol. III: Viruses and Viroids, pp. 6585. New York: Pergamon. Con RS and Coutts RHA (1993) The closteroviruses, capilloviruses and other similar viruses: a short review. Journal of General Virology 74: 14751483. Dolja VV, Karasev AV and Koonin EV (1994) Molecular biology and evolution of closteroviruses: sophisticated build-up of large RNA genomes. Annual Review of Phytopathology 32: 261285. Francki RIB, Milne RG and Hatta T (1985) Closteroviruses. In: Atlas of Plant Viruses, vol. 2, p. 219. Boca Raton, FL: CRC Press. German-Retana S, Candresse T and Martelli GP (1999) Closteroviruses. In: Webster RG and Grano A (eds), Encyclopedia of Virology, 2nd edn. New York: Academic Press (in press).

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Lister RM and Bar-Joseph M (1981) Closteroviruses. In: Kurstak E (ed.), Handbook of Plant Virus Infections and Comparative Diagnosis, pp. 810844. Amsterdam: Elsevier/North Holland Biomedical Press.

Martelli GP, Saldarelli P and Boscia D (1997) Filamentous viruses of the grapevine: Closteroviruses. In: Monette PL (ed), Filamentous Viruses of Woody Plants, pp. 19. Trivandrum: Research Signpost.

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