Constriction of endoplasmic reticulum tubules by the viral movement protein BMB2 is associated with local BMB2 anchorage at constriction sites.

Plant virus-encoded movement proteins (MPs) interact with endoplasmic reticulum (ER) membranes, the cytoskeleton, and plasmodesmata (PD) to mediate intracellular delivery of the virus genome to PD and its further transport through PD from infected to healthy cells. The Hibiscus green spot virus MP termed BMB2 has been shown to induce constrictions of ER tubules and to occur at highly curved membranes, thus showing properties similar to those of reticulons, a class of cellular proteins inducing membrane curvature and shaping the ER tubules. Consistent with this BMB2 function, mRFP-BMB2 localizes to discrete, constricted regions scattered along the ER tubules. Here, using BMB2-mRFP fusion protein as a BMB2 derivative with partially disabled functionality, we demonstrate that the focal localization of BMB2 to discrete sites along the ER tubules is insufficient to induce local tubule constrictions at these sites, suggesting that the formation of ER tubule constrictions represents a specific BMB2 function and is not simply a mechanistic consequence of its localization to the ER. The presented data suggest that the formation of ER-residing BMB2-containing distinct small aggregates, or protein platforms, can be uncoupled from BMB2-induced ER tubule constrictions, whereas the anchoring of platforms at particular ER sites appears to be linked to the constriction of ER tubules at these sites.

Cell-to-cell movement and assembly of a plant closterovirus: roles for the capsid proteins and Hsp70 homolog.

Diverse animal and plant viruses are able to translocate their virions between neighboring cells via intercellular connections. In this work, we analyze the virion assembly and cell-to-cell movement of a plant closterovirus and reveal a strong correlation between these two processes. The filamentous virions of a closterovirus possess a long body formed by the major capsid protein (CP) and a short tail formed by the minor capsid protein (CPm). Genetic and biochemical analyses show that the functions of these virion components are distinct. A virion body is required primarily for genome protection, whereas a tail represents a specialized device for cell-to-cell movement. Furthermore, tail assembly is mediated by the viral Hsp70 homolog (Hsp70h) that becomes an integral part of the virion. Inactivation of the ATPase domain of Hsp70h results in assembly of tailless virions that are incapable of translocation. A dual role for the viral molecular chaperone Hsp70h in virion assembly and transport, combined with the previous finding of this protein in intercellular channels, allowed us to propose a model of closteroviral movement from cell to cell.

Functional specialization and evolution of leader proteinases in the family Closteroviridae.

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

Leader proteinase of the beet yellows closterovirus: mutation analysis of the function in genome amplification.

The beet yellows closterovirus leader proteinase (L-Pro) possesses a C-terminal proteinase domain and a nonproteolytic N-terminal domain. It was found that although L-Pro is not essential for basal-level replication, deletion of its N-terminal domain resulted in a 1, 000-fold reduction in RNA accumulation. Mutagenic analysis of the N-terminal domain revealed its structural flexibility except for the 54-codon-long, 5'-terminal element in the corresponding open reading frame that is critical for efficient RNA amplification at both RNA and protein levels.

Interaction between HSP70 homolog and filamentous virions of the Beet yellows virus.

An HSP70 homolog (HSP70h), encoded by the Closterovirus Beet yellows virus (BYV), functions in viral movement from cell to cell. A previous study revealed that in infected cells, HSP70h colocalizes with the masses of BYV filamentous virions. Here we demonstrate that HSP70h forms a physical complex with BYV virions. This conclusion is based on both the comigration of HSP70h with BYV virions in sucrose density gradients and the coimmunoprecipitation of the HSP70h and BYV capsid protein using anti-HSP70h serum. The HSP70h-virion complex is stable at high concentrations of sodium chloride; its dissociation using sodium dodecyl sulfate, lithium chloride, or alkaline pH was accompanied by virion disassembly. However, the complex formation does not involve covalent bonds between HSP70h and virion components. Each BYV virion contains approximately 10 molecules of HSP70h. The possible role of HSP70h interaction with the virions in cell-to-cell movement of BYV is discussed.

Genetic analysis of the cell-to-cell movement of beet yellows closterovirus.

A beet yellows closterovirus (BYV) variant expressing green fluorescent protein and leaves of BYV local lesion host Claytonia perfoliata were used to reveal genetic requirements for BYV cell-to-cell movement in leaf epidermis and mesophyll. A series of mutations targeting genes that are not involved in amplification of the viral positive-strand RNA was analyzed. The products of genes coding for a 6-kDa hydrophobic protein (p6) and a 64-kDa protein (p64), as well as for minor and major capsid proteins, were found to be essential for intercellular translocation of BYV. In a previous work, we have demonstrated that the BYV HSP70-homolog (HSP70h) also plays a critical role in viral movement (V. V. Peremyslov, Y. Hagiwara, and V. V. Dolja, 1999, Proc. Natl. Acad. Sci. USA, 96, 14771-14776). Altogether, a unique protein quintet including three dedicated movement proteins (p6, p64, and HSP70h) and two structural proteins is required to potentiate the cell-to-cell movement of a closterovirus. The corresponding BYV genes are clustered in a block that is conserved among diverse representatives of the family Closteroviridae.

HSP70 homolog functions in cell-to-cell movement of a plant virus.

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

Regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization.

A reporter open reading frame (ORF) coding for a fusion of bacterial beta-glucuronidase (GUS) with a proteinase domain (Pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (BYV). Insertion of this reporter ORF between the first and second codons of the BYV ORFs encoding the HSP70 homolog (HSP70h), a major capsid protein (CP), and a 20-kDa protein (p20) resulted in the expression of the processed GUS-Pro reporter from corresponding subgenomic RNAs. The high sensitivity of GUS assays permitted temporal analysis of reporter accumulation, revealing early expression from the HSP70h promoter, followed by the CP promoter and later the p20 promoter. The kinetics of transcription of the remaining BYV genes encoding a 64-kDa protein (p64), a minor capsid protein (CPm), and a 21-kDa protein (p21) were examined via Northern blot analysis. Taken together, the data indicated that the temporal regulation of BYV gene expression includes early (HSP70h, CPm, CP, and p21 promoters) and late (p64 and p20 promoters) phases. It was also demonstrated that the deletion of six viral genes that are nonessential for RNA amplification resulted in a dramatic increase in the level of transcription from one of the two remaining subgenomic promoters. Comparison with other positive-strand RNA viruses producing multiple subgenomic RNAs showed the uniqueness of the pattern of closterovirus transcriptional regulation.

Subcellular localization of the HSP70-homolog encoded by beet yellows closterovirus.

Closteroviridae is the only viral family coding for a homolog of HSP70 (HSP70h). Polyclonal antiserum to recombinant beet yellows closterovirus (BYV) HSP70h was generated and used for immunogold labeling of the leaf samples derived from the infected Nicotiana benthamiana plants. Ultrastructural analysis revealed the preferential accumulation of BYV in phloem, although occasional infection of the leaf mesophyll cells was also observed. The strongest HSP70h-specific labeling was associated with virion aggregates and vesicles harboring scattered virions. HSP70h was also observed in close proximity of plasmodesmata and inside the plasmodesmatal channels. The possible role of the BYV HSP70h in RNA encapsidation was tested in tobacco protoplasts. A BYV mutant possessing an inactivated HSP70h gene exhibited no detectable encapsidation defects. Collectively, the obtained results suggested that closteroviral HSP70h escorts the virions to their destinations inside the infected cells and possibly participates in the intercellular translocation of BYV.

An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication.

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of approximately 20 kilobases that expresses the replicase-associated genes as an approximately 400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.

Histidine-tagging and purification of tobacco etch potyvirus helper component protein.

The coding sequence for a series of six histidines (his-tag) was inserted near the 5' terminus of the helper component (HC) coding region of tobacco etch potyvirus (TEV). Full length genomic clones containing the his-tag coding sequence were infectious and produced symptoms in tobacco (Nicotiana tabacuma) similar to those induced by wild-type TEV. The modified virus was genetically stable and the his-tag sequence was maintained through at least four cycles of aphid transmission. A protocol for purifying rapidly the HC protein, based on the affinity of its his-tag for Ni(2+)-charged resin, yielded large amounts of his-tagged HC protein that was fully functional as demonstrated by aphid transmission experiments.

Genes required for replication of the 15.5-kilobase RNA genome of a plant closterovirus.

A full-length cDNA clone of beet yellows closterovirus (BYV) was engineered and used to map functions involved in the replication of the viral RNA genome and subgenomic RNA formation. Among 10 open reading frames (ORFs) present in BYV, ORFs 1a and 1b suffice for RNA replication and transcription. The proteins encoded in these ORFs harbor putative methyltransferase, RNA helicase, and RNA polymerase domains common to Sindbis virus-like viruses and a large interdomain region that is unique to closteroviruses. The papain-like leader proteinase (L-Pro) encoded in the 5'-proximal region of ORF 1a was found to have a dual function in genome amplification. First, the autocatalytic cleavage between L-Pro and the remainder of the ORF 1a product was essential for replication of RNA. Second, an additional L-Pro function that was separable from proteolytic activity was required for efficient RNA accumulation. The deletion of a large, approximately 5.6-kb, 3'-terminal region coding for a 6-kDa hydrophobic protein, an HSP70 homolog, a 64-kDa protein, minor and major capsid proteins, a 20-kDa protein, and a 21-kDa protein (p21) resulted in replication-competent RNA. However, examination of mutants with replacements of start codons in each of these seven 3'-terminal ORFs revealed that p21 functions as an enhancer of genome amplification. The intriguing analogies between the genome organization and replicational requirements of plant closteroviruses and animal coronavirus-like viruses are discussed.

Mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets.

Mutations of K --> E in the highly conserved 'KITC' motif of the potyvirus helper component (HC) protein result in loss of HC function in aphid transmission, presumably because of inability to interact with virions, stylets or both. In this study we show that HC of potato virus C (PVC), a naturally occurring variant of potato virus Y (PVY) that has the K --> E mutation, lacks the ability to be retained in stylets, whereas PVY HC is retained. The K --> E mutation in either PVC or a site-directed mutant of tobacco etch virus (TEV) did not hinder binding to capsid protein, nor did deletion of the N-terminal 107 aa of TEV HC. An additional mutation, F -->, L at aa 10 of TEV HC, which renders HC non-functional but does not affect binding to capsid protein, is reported. Collectively, the results suggest that the N-terminal domain is required for interaction of HC with stylets rather than for binding to virions.

Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors.

A system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterovirus (BYV). Approximately 4 micrograms/g of 20-kDa BYV protein was readily isolated from plants systemically infected by hybrid TEV. In contrast, only minute quantities of 22-kDa BYV capsid protein (CP) histidine-tagged at its N or C terminus could be purified. Rapid degradation of the recombinant CP has been implicated in its failure to accumulate in infected plants. Fusion with TEV HC-Pro stabilized the histidine-tagged BYV CP and facilitated purification of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant proteins were recognized by histidine-tag-specific monoclonal antibody in immunoblot analysis. These results demonstrate the utility of a designed series of TEV vectors for expression, detection, and purification of the recombinant proteins and suggest that intrinsic protein stability is a major factor in a recovery of recombinant proteins from plants.

Suppression of potyvirus infection by coexpressed closterovirus protein.

A tobacco etch virus (TEV)-based expression vector has been used for insertion of several ORFs derived from the unrelated beet yellows virus (BYV). Hybrid TEV variants expressing the BYV capsid protein, 20-kDa protein, or HSP70 homolog systemically infected Nicotiana tabacum and stably retained BYV sequences. In contrast, insertion of the ORF encoding BYV leader proteinase (L-Pro) resulted in severely impaired systemic transport and accumulation of recombinant TEV. Progeny of this virus underwent various deletions affecting the L-Pro sequence and mitigating the defects in virus spread. Model experiments involving several spontaneous and engineered mutants indicated that the central domain of BYV L-Pro was responsible for the defect in hybrid virus accumulation, whereas full-size L-Pro was required for maximal debilitation of systemic transport. Strikingly, BYV L-Pro expression did not debilitate systemic infection of hybrid TEV in Nicotiana benthamiana plants. No major defects in replication or encapsidation of recombinant RNA were revealed in N. tabacum protoplasts. These results indicated that BYV L-Pro specifically interfered with TEV systemic transport and accumulation in a host-dependent manner and suggested a potential utility of closterovirus L-Pro as an inhibitor of potyvirus infection. In addition, it was demonstrated that the 107-amino-acid-residues-long N-terminal part of the TEV helper component proteinase is not essential for systemic infection.

Roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element.

The roles of the capsid protein (CP) and the CP coding sequence of tobacco etch potyvirus (TEV) in genome amplification were analyzed. A series of frameshift-stop codon mutations that interrupted translation of the CP coding sequence at various positions were introduced into the TEV genome. A series of 3' deletion mutants that lacked the CP coding sequence beyond each of the frameshift-stop codon mutations were also produced. In addition, a series of 5' CP deletion mutants were generated. Amplification of genomes containing either frameshift-stop codon insertions after codons 1, 59, 103, and 138 or genomes containing the corresponding 3' deletions of the CP coding sequence was reduced by 100- to 1,000-fold relative to that of the parental genome in inoculated protoplasts. In contrast, a mutant containing a frameshift-stop codon after CP position 189 was amplified to 27% of the level of the parental virus, but the corresponding 3' deletion mutant lacking codons 190 to 261 was nonviable. Deletion mutants lacking CP codons 2 to 100, 2 to 150, 2 to 189, and 2 to 210 were amplified relatively efficiently in protoplasts, but a deletion mutant lacking codons 2 to 230 was nonviable. None of the amplification-defective frameshift-stop codon or deletion mutants was rescued in transgenic cells expressing TEV CP, although the transgenic CP was able to rescue intercellular movement defects of replication-competent CP mutants. Coupled with previous results, these data led to the conclusions that (i) TEV genome amplification requires translation to a position between CP codons 138 and 189 but does not require the CP product and (ii) the TEV CP coding sequence contains a cis-active RNA element between codons 211 and 246. The implications of these findings on mechanisms of RNA replication and genome evolution are discussed.

Capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus.

The tobacco etch potyvirus (TEV) capsid protein (CP) is necessary for cell-to-cell and long distance transport of the virus in plants. In this study, the transport phenotypes of TEV mutants containing CPs with a substitution of the highly conserved Ser122 (termed S122W) within the core domain, or with a deletion of sequences encoding 17 amino acid residues comprising most of the variable C-terminal domain (delta C), were analyzed. The S122W and delta C mutant genomes were amplified to levels comparable to parental virus in protoplasts. The S122W mutant was encapsidation-defective, although in transgenic plants expressing wild-type CP a small number of virions were observed after prolonged incubation. Cells infected by the delta C mutant produced virions, indicating that the C-terminal domain is not necessary for encapsidation. The mutants exhibited unique defects in cell-to-cell and long distance movement in plants. The S122W mutant was confined to single, primarily inoculated epidermal cells in nontransgenic plants, but the cell-to-cell movement defect was rescued efficiently by transgenic CP. Long distance movement of this mutant was also rescued in transgenic plants, but accumulation in systemically infected tissue was low compared to parental virus. The delta C mutant exhibited a slow cell-to-cell movement phenotype in inoculated leaves and a complete inability to move systemically in nontransgenic plants. Transgenic CP was able to rescue partially the slow cell-to-cell movement defect of the delta C mutant, but not the long distance transport defect. Taken together with previous results, these data suggest that the core domain of TEV CP provides a function essential during cell-to-cell movement and that the variable N- and C-terminal regions exposed on the virion surface are necessary for long distance transport. In addition, trans-inhibition models are presented to account for the widely differing transgenic complementation efficiencies of the various movement-defective mutants.

Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.