Virus Rearch117(2006)
38–51
Comparative and functional genomics of closterovirus
Valerian V.Dolja a,∗,Jan F.Kreuze b,c,Jari P.T.Valkonen c,d
a Department of Botany and Plant Pathology and Center for Genome Rearch and Biocomputing,Oregon State University,Corvallis,OR97331,USA
b Germplasm Enhancement and Crop Improvement Division,International Potato Center(CIP),Apartado1558,Lima12,Peru
口言c Department of Plant Biology an
d Forest Genetics,Swedish University of Agricultural Sciences,SE-75007Uppsala,Sweden
d Department of Applied Biology,University of Helsinki,FIN-00014,Finland
Abstract
接亲保证书The largest extant RNA genomes are found in two diver families of positive-strand RNA virus,the animal Coronaviridae and the plant Closteroviridae.Comparative analysis of the virus fr
遵义市第十三中学
om the latter family reveals three levels of gene conrvation.The most conrved gene module defines RNA replication and is shared with plant and animal virus in the alphavirus-like superfamily.A module offive genes that function in particle asmbly and transport is a hallmark of the family Closteroviridae and was likely prent in the ancestor of all three closterovirus genera. This module includes a homologue of Hsp70molecular chaperones and three diverged copies of the capsid protein gene.The remaining genes show dramatic variation in their numbers,functions,and origins among closterovirus within and between the genera.Proteins encoded by the genes include suppressors of RNA silencing,RNA III,papain-like proteas,the AlkB domain implicated in RNA repair,Zn-ribbon-containing protein,and a variety of proteins with no detectable homologues in the current databas.The evolutionary process that have shaped the complex andfluid genomes of the large RNA virus might be similar to tho that have been involved in evolution of genomic complexity in other divisions of life.
©2006Elvier B.V.All rights rerved.
Keywords:Virus evolution;Closteroviridae;Closterovirus;Crinivirus;Ampelovirus
1.Introduction
With their20–30kb genomes,the largest known RNA virus are no match for the champions of the DNA virus world who genomes reach1.2Mb(Iyer et al.,2006),let alone cellular life forms.Nevertheless,RNA virus play a prominent role in our understanding of life’s origin and evolution.Becau RNA is widely believed to predate DNA as the genetic mate-rial,RNA virus could be living fossils of the primordial RNA world(Joyce,2002;Koonin and Martin,2005).There-fore,at least some genes of RNA virus are likely to encode extremely ancient,perhaps,primeval proteins involved in repli-cation and metabolism of nucleic acids.From a somewhat different,comparative-genomic perspective,large RNA virus provide an opportunity to investigate problems of genome com-plexity and its evolution on a relatively modest,tractable scale.
Closterovirus share a conrved core of genes involved in replication with other animal and plant virus within the
宁夏银川∗Corresponding author.Tel.:+15417375472;fax:+15417373573.
E-mail address:state.edu(V.V.Dolja).alphavirus-like superfamily of positive-strand RNA virus (Dolja et al.,1994).However,closterovirus stand alone in regard to their genetic capacity and variability,as well as the unique morphology of their particles.It is instructive to com-par
e the genera Closterovirus and Tobamovirus that both belong to the alphavirus-like superfamily and share the helical virion architecture(Fig.1).All tobamovirus have∼6.5kb genomes that code for four proteins,one of which asmbles to form the rod-shaped virion(Fig.1B and C).In contrast,the size of closterovirus genomes varies from∼15.5to∼19.5kb with a coding capacity of10–14proteins(Figs.1A,B and2).Filamen-tous virions of closterovirus incorporate at leastfive proteins that are asmbled into a long body of uniform morphology and a short gmented tail(Fig.1C).Thus,the larger amount of genetic material in closterovirus translates into the incread structural complexity and genetic variation among individual virus.
Here we explore the gene repertoire of the family Clos-teroviridae in an attempt to reconstruct the evolutionary history of the virus.We also u the available information on the gene functions and regulation to propo a working model of the infection cycle for a‘generic’closterovirus.
0168-1702/$–e front matter©2006Elvier B.V.All rights rerved. doi:10.1016/j.virusres.2006.02.002
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Fig.1.Comparison of the genetic and structural complexity of Beet yellows virus(BYV),genus Closterovirus,and Tobacco mosaic virus(TMV),genus Tobamovirus.深圳荔园小学
(A)Genome map,functions,and evolutionary connections of BYV.The ORFs are shown as cylinders with associated protein designations.L-Pro,leader proteina; MET,HEL,and POL,methyltransfera,RNA helica,and RNA-dependent RNA polymera domains of the replica,respectively;p6,a6-kDa protein;Hsp70h, a Hsp70-homologue;p64,a64-kDa protein;CPm and CP,the minor and major capsid proteins,respectively;p20and p21,the20and21-kDa proteins,respectively. The quence similarity between CP,CPm,and the C-terminal domain of p64is illustrated with the same color.The protein functions are indicated above and below the diagram,while the evolutionary conrvation patterns are shown at the bottom.(B)Genome map,functions,and evolutionary connections of TMV.P30,a30-kDa protein.Other designations are the same as in(A).(C)Cartoons of the TMV and BYV virions.The TMV virions are rigid helical rods of∼300nm in length and 18nm in diameter.The BYV virions areflexuous,helicalfilaments of∼1400nm in length and12nm in diameter with the∼100nm-long tails that are∼8nm in diameter.The protein composition and the length of the encapsidated viral RNA is shown.
2.Replication-related genes
As is the ca for all positive-strand RNA virus,putative components of the closteroviral RNA replica are expresd directly from the virion RNA(Karav et al.,1989).The prod-uct of the5 -terminal open reading frame(ORF)contains RNA methyltransfera(MET)and RNA helica(HEL)domains. The RNA-dependent RNA polymera(POL)is encoded by a downstream ORF that is presumably expresd via a+1 translational frameshift(Agranovsky et al.,1994;Karav et al.,1995).Thus,translation of the genomic RNA results in two large polyproteins,one spanning MET–HEL,and the other one encompassing MET–HEL–POL;this cond,larger polyprotein is produced in much smaller quantities due to the low frequency of frameshifting.The MET–HEL–POL mod-ule of closterovirus is universally conrved in the entire alphavirus-like superfamily(Fig.1A and B)(Koonin and Dolja, 1993).
A peculiar feature of the closteroviral replicas is the pres-ence of a large variable region between the MET and HEL domains.This region is procesd by an unknown mecha-nism to produce parate MET-and HEL-containing products (Erokhina et al.,2000).Becau ectopic expression of the MET–HEL region in plants is sufficient to generate the p-arate products,the processing could involve either a cryptic proteolytic activity within the MET–HEL polyprotein itlf, or a cellular pr
otea(Peremyslov and Dolja,unpublished data).
In addition to their enzymatic functions,RNA replicas of alphavirus-like virus direct asmbly of the replication com-partments.The principal component of the compartments is a protein shell formed by the MET–HEL protein subunits (Schwartz et al.,2002).This shell is enveloped by a mem-brane derived from the endoplasmic reticulum(ER)or other endomembrane.In accord with this paradigm,we found that closterovirus infection results in a drastic remodeling of the ER network that is transformed into a large vesicular factory of viral RNA.We also found that transient expression of the MET–HEL polyprotein alone is sufficient to restructure ER(Prokhnevsky et al.,unpublished data).As propod by Schwartz et al. (2002),one or a few POL-containing replication complexes are prent within each virus-induced vesicle to amplify the viral genome and to produce subgenomic mesnger RNA (sgRNAs).
40V .V .Dolja et al./Virus Rearch 117(2006)
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Fig.2.Genome maps of the lected reprentatives of the family Closteroviridae .The genera names are shown at the left.BYV ,Beet yellows virus (Agranovsky et al.,1994);CTV ,Citrus tristeza virus (Karav et al.,1995);LIYV ,Lettuce infectious yellows virus (Klaasn et al.,1995);SPCSV ,Sweet potato chlorotic stunt virus (Kreuze et al.,2002);GLRaV-3,Grapevine leafroll-associated virus-3(Ling et al.,2004).The designations and color code for the conrved proteins are the same as in Fig.1A.L1and L2,tandem of the leader proteinas in CTV .The unique proteins in each genome are shown in colors as dissimilar as possible with their approximate molecular weight following common ‘p’designator,except for RNA III and AlkB domain (e the text for further information).猫咪可以吃狗粮吗
The extreme 5 -terminal region of the closterovirus RNA encodes the papain-like leader proteina (L-Pro)that is relead from the polyprotein via autoprocessing (Agranovsky et al.,1994).Even though papain-like proteinas are common among alphavirus-like virus,their typical role in the viral life cycle is distinct from that of L-Pro.The papain-like proteas of alphavirus,rubivirus,or tymovirus are located between the replica domains and are responsible for multiple process-ing events within the polyprotein (Dougherty and Semler,1993).In contrast,the closterovirus L-Pro is located upstream of the replica polyprotein and is not required for its processing (e above).
In addition to autocatalytic processing,L-Pro plays a promi-nent role in the amplification of the viral g
enome:elimination of L-Pro reduces the amount of viral RNA to ∼0.1%of the wild-type level (Peng and Dolja,2000).Becau L-Pro co-localizes with the replication vesicles (Zinovkin et al.,2003),its function in RNA amplification could involve either activation of the viral replica or protection of the RNA from degradation by a host defen system.
Some closterovirus posss a tandem of the leader pro-teinas that probably evolved via gene duplication.As was found using the gene swapping approach,one of the tandem proteinas (L1),but not the other (L2),can functionally substi-tute for a single L-Pro by enhancing virus RNA amplification (Peng et al.,2001).When expresd together,L1and L2act流产后腰疼
synergistically to provide for even higher levels of viral RNA.Interestingly,proliferation of the papain-like leader proteinas is also obrved in some virus of the order Nidovirales that includes the family Coronaviridae (Gorbalenya et al.,2006).This evolutionary convergence might reflect common require-ments pod by the increa in the size of the viral genomes and encoded polyproteins (Peng et al.,2002).
Another feature shared by closterovirus and nidovirus is expression of the multiple internal genes via a nested t of the 3 -coterminal subgenomic (sg)RNAs.Usually,the sgRNAs are functio
nally monocistronic,each expressing only one protein from the 5 -proximal ORF.While many diver positive-strand virus produce from one to three subgenomic sgRNAs (Miller and Koev,2000),nidovirus make up to 9,and closterovirus up to 10sgRNAs.Although the details of transcription mech-anisms may vary among the virus of the order Nidovirales ,they share certain important features (Gorbalenya et al.,2006).The short,AU-rich,transcription-regulating quences (TRS)are prent upstream from the 5 -terminal and all internal ORFs.The TRSs are thought to mediate termination of the minus-strand synthesis to produce templates for the positive-strand sgRNAs.In arterivirus and coronavirus,termination at the TRS of each internal ORF is followed by the template ‘jumping’such that transcription complex lands to 5 -proximal TRS and copies the part of the 5 -leader region.This discontinuous transcrip-tion mode resulting in a mosaic structure of the sgRNAs is a
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unique and remarkable feature of the nidovirus genome expres-sion mechanism(Gorbalenya et al.,2006;Pasternak et al.,2001; Sawicki et al.,2001;Zuniga et al.,2004).
The closterovirus do not employ discontinuous transcrip-tion and have no TRS-like,common elem
ent in the regions that direct sgRNAs synthesis(Karav et al.,1997;Kreuze et al., 2002;Peremyslov and Dolja,2002).It is not known if,simi-lar to smaller alpha-like virus,closteroviral sgRNA synthesis involves internal initiation using genome-size,negative-strand template(Miller and Koev,2000).In fact,occurrence of the minus strands of sgRNAs in closterovirus-infected cells(Dolja et al.,1990;Hilf et al.,1995)is better compatible with the nidovirus-like,minus-strand termination model.Additional sup-port for this model comes from extensive analysis of the CTV-specific RNA species.It was found that the controller elements located upstream from each internal CTV ORF direct formation of not only the positive and negative strands of sgRNAs,but also a t of the5 -coterminal positive-strand RNAs of subge-nomic size(Gowda et al.,2001).The latter RNAs terminate at variable positions upstream of the initiation site of the corre-sponding sgRNAs.The simplest reconciliation of the data is that the sgRNA controller elements,which contain hairpin struc-tures,act as terminators during transcription of both positive, and negative strands of the viral RNA.Although there were no reports of two distinct ts of subgenomic RNAs in nidovirus, this issue ems to derve more experimental scrutiny.
Although the functional significance,if any,of the5 -coterminal,subgenomic-size,CTV RNAs is unknown,one may wonder if coexistence in the infected cells of the multitude of RNA species may pr
omote formation of the recombinant RNA molecules.Indeed,plants infected with closterovirus often contain defective RNAs(dRNAs),some of which appear to result from recombination between sgRNAs and their coun-terparts from the5 -terminal region(Che et al.,2002;Rubio et al.,2000).Moreover,CTV dRNAs reminiscent of crinivirus genomic RNAs1and2were described(Che et al.,2003)sug-gesting that the sgRNAs may facilitate closterovirus evolution, be it genome gmentation obrved in crinivirus(Klaasn et al.,1995;Liveratos et al.,2004),gene duplication(Boyko et al.,1992;Napuli et al.,2003),or recombinational accretion of the viral or cellular genes(Agranovsky et al.,1991;Kreuze et al.,2002).
Even though sgRNA controller elements of closterovirus exhibit little or no quence conrvation within the individual virus genomes or between the related closterovirus(Gowda et al.,2001;Kreuze et al.,2002;Peremyslov and Dolja,2002),they likely posss common features of the higher order structures. The elements function efficiently in a heterologous genetic ,they are recognized by heterologous repli-cas(Peremyslov et al.,1999).The structural variability of the sgRNA controllers ems to contribute to the regulation of the level and timing of viral gene expression(Hagiwara et al., 1999;Hilf et al.,1995).On the more practical side,the promis-cuity of the RNA replicas toward sgRNA controllers facilitates utilization of closterovirus as versatile gene ex
pression vec-tors.The vectors can accommodate multiple expression cas-ttes and efficiently produce reporters or other beneficial pro-teins in the infected plants(Peremyslov and Dolja,unpublished data).
有的放矢It ems that the most intriguing questions pertinent to clos-terovirus RNA synthesis are the transcription mechanisms,the enigmatic function of the large,variable central domain of the viral replica,and the mechanism by which L-Pro enhances RNA amplification.The answers to the questions will cer-tainly contribute to our understanding of what does it take to replicate and express large infectious RNAs.
3.Quintuple gene block
The replication gene block of closterovirus is rank and file of the alphavirus-like superfamily,some deviations from the prototype notwithstanding.In contrast,thefive downstream genes compri a truly unique genetic module not found outside the family Closteroviridae(Dolja et al.,1994).This quintuple gene block(QGB)codes for a∼6-kDa hydrophobic protein (p6),an Hsp70homologue(Hsp70h),a∼60-kDa protein(p60), the minor capsid protein(CPm),and the major capsid protein (CP)(Fig.1A).P6is a single-span transmembrane protein that resides in ER and functions in virus movement from cell to c
ell (Alzhanova et al.,2000;Peremyslov et al.,2004b).Becau p6 is not required for virus replication or asmbly(Alzhanova et al.,2001;Peremyslov et al.,1998),it can be considered a con-ventional movement protein(MP).
CP forms a long,helical body of theflexuous,filamentous virions,and encapsidates∼95%of the viral RNA(Fig.1C). CPm is paralogous to CP and functions as a principal com-ponent of the short virion tail(Agranovsky et al.,1995;Tian et al.,1999).In addition to CPm,the tail asmbly requires Hsp70h and p60,each of which is also an integral,although a minor tail component(Alzhanova et al.,2001;Napuli et al.,2000,2003;Peremyslov et al.,2004a;Satyanarayana et al.,2000,2004).Similar to cellular molecular chaperones of Hsp70family,closteroviral Hsp70h posss a highly conrved, N-terminal,ATPa domain and a less conrved C-terminal domain(Agranovsky et al.,1991;Bork et al.,1992).Extensive mutation analysis revealed that each of the domains is inti-mately involved in the virion asmbly(Alzhanova et al.,2001; Prokhnevsky et al.,unpublished data).It was also found that p60 posss the CP-like,virion-embedded,C-terminal domain,and the N-terminal domain,which is expod at the virion’s surface (Napuli et al.,2003).Hsp70h and p60act cooperatively to facil-itate incorporation of CPm and to define the proper tail length (Satyanarayana et al.,2004;Alzhanova et al.,unpublished data). The tail encapsidates the5 -terminal,∼700nt-long,RNA region (Fig.1C)that cont
ains a packaging signal recognized by CPm (Peremyslov et al.,2004a;Satyanarayana et al.,2004).Interest-ingly,virion bodies and tails can be asmbled independently of each other.
Atomic force microscopy of Beet yellows virus(BYV,genus Closterovirus)virions showed that the tails are narrower than the bodies and exhibit three-gment structure(Peremyslov et al., 2004a).A pointed tip gment of the BYV tails contains a20-kDa protein(p20)(Fig.1C)that is dispensable for incorporation of other virion components.Inactivation of p20results in shorter
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tails and apparently normal bodies.Becau the orthologs of BYV p20are not found in other closterovirus,it is not clear if the tail gmentation pattern is a common feature of the family.
A salient functional aspect of the BYV virions is that each of thefive structural proteins is also required for virus trans-port(Dolja,2003).In the ca of CP,this requirement could stem,simply,from the need to protect the long and degradation-prone viral RNA during its transport.Indeed,the cell-to-cell movement cycle of a closterovirus takes a day compared to two hours for TMV.However,it ems unlikely that the transport-related function of the tail is merely protective.First,CP can encapsidate the entire genome if the tail formation is prevented by mutations(Alzhanova et al.,2001).
Second,the tail har-bors Hsp70h,the only viral protein that was found in plas-modesmata(PD),intercellular channels through which virus translocate(Medina et al.,1999).Third,p20is dispensable for virion asmbly and cell-to-cell movement,but is required for long-distance transport of BYV through the vascular system (Prokhnevsky et al.,2002).All this prompted interpretation of the tail as a device that evolved to facilitate cell-to-cell and systemic transport of the large closterovirus genomes(Dolja, 2003).It should be noted that this concept awaits experimen-tal paration of asmbly and transport functions for at least some tail proteins unless there is a causal relationship between the two.
Even though the QGB genes are universally conrved within the family,their order is not.In the genera Ampelovirus and Crinivirus,the order of CPm and CP is reverd,and the CPm is much larger than in the genus Closterovirus(Fig.2)(Klaasn et al.,1995;Kreuze et al.,2002;Ling et al.,2004;Melzer et al., 2001).Other examples of QGB variation include duplication and divergence of the CPm gene in Grapevine leafroll-associated virus-1(Fazeli and Rezaian,2000),reshuffling of QGB in Little cherry virus(Theilmann et al.,2002),and occurrence of enig-matic additional ORFs within QGB of crinivirus and certain ampelovirus.
4.Suppressors of RNA silencing
It has been long accepted that all non-defective plant virus must code for at least three functions:genome replication,encap-sidation,and transport within infected plants.Arguably,suppres-sion of RNA silencing has emerged recently as a fourth universal function encoded in plant virus(Baulcombe,2004).Some plant virus posss dedicated suppressors,while others del-egate this function to replicational,structural,or transport pro-teins(Silhavy and Burgyan,2004;V oinett,2005).Interestingly, most of the dedicated viral suppressors reprent small pro-tein families without detectable homologous relationship to any other viral or host proteins.This tendency is often taken as evi-dence of relatively recent origins of the suppressors that evolved to counteract a powerful system of host defen against para-sitic RNAs.The major components of RNA silencing machinery are conrved between diver eukaryotes suggesting that this machinery emerged not much later than eukaryotes themlves (Anantharaman et al.,2002;Zamore and Haley,2005).Search for RNA silencing suppressors in closterovirus yielded v-eralfindings that further highlighted the trends in suppressor evolution.
Screening of the BYV genome for silencing suppressor func-tions showed that the21-kDa protein(p21)is a suppressor of RNA silencing and that the orthologous proteins from other virus of the genus Closterovirus also posss suppressor activ-ity(Chiba et al.,2006;Reed et al.,2003).It has
been found that p21specifically binds double-stranded forms of the small inter-fering RNA(siRNA)or microRNA(miRNA)(Chapman et al., 2004).Thus,the molecular mechanism of p21action is simi-lar to that of another well-studied suppressor,p19of Tomato bushy stunt virus(Silhavy and Burgyan,2004):both suppres-sors quester small RNA effectors of silencing and prevent their loading into the RNA-Induced Silencing Complex(RISC). However,the suppressors cannot discriminate between siR-NAs that target viral genomes and endogenous siRNAs and miRNAs that are involved in regulation of plant development.As a result,accumulation of p21or p19in plants induces develop-mental abnormalities many of which are identical to symptoms of viral infection(Chapman et al.,2004).It was concluded that,at least in part,viral pathogenicity is due to interference of silencing suppressors with developmental function of plant small RNAs.Despite their mechanistic similarity,p21and p19 appear to be structurally and evolutionarily unrelated and neither has detectable homologues outside the respective virus genera (Vargason et al.,2003;Ye and Patel,2005).
Although Citrus tristeza virus(CTV)encodes p20,a p21-like suppressor of RNA silencing,screening of the CTV genome revealed an additional suppressor,p23,that has no homologues in other closterovirus(Fig.2)(Lu et al.,2004).Subquent computer analysis of the p23quence showed th
at it has a spe-cific form of the Zn-ribbon module that is prent in a variety of cellular proteins and also in veral proteins encoded by virus of the genera Carlavirus and Vitivirus(Chiba et al.,2006).It ems likely that the latter viral proteins are distant homologues of p23.Should it be demonstrated that the proteins are sup-pressors of RNA silencing,this will be thefirst viral suppressor family reprented in diver virus and,possibly,derived from a common host ancestor.It was also found that both CTV p20 and CP can interfere with the systemic spread of silencing,while p23can only suppress the local silencing(Lu et al.,2004).Thus, CTV evolved a complex system of RNA silencing suppression with three components targeting distinct facets of RNA silencing respon.
Another remarkable discovery in the area of closterovirus counter-defen is a two-component RNA silencing suppression system in Sweet potato chlorotic stunt virus(SPCSV)(Kreuze et al.,2005).One of the components of this system is a22-kDa protein(p22)that posss suppressor activity on its own (Fig.2).This protein is unique to SPCSV and has no detectable quence similarity to other proteins.A cond component is an RNa III homologue containing both the endonuclea and the dsRNA-binding activities typically found in bonafide cel-lular RNas III including Dicer and Dicer-like proteins,which are involved in the generation and functioning of siRNAs and miRNAs.In addition
to SPCSV,RNa III is encoded by veral nucleocytoplasmic large DNA virus(Iyer et al.,2006)where
