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At least 19 recordsLinked to original sources
PubMed2004

Little cherry virus-2: sequence and genomic organization of an unusual member of the Closteroviridae.

The complete genomic sequence of variant USA6b of Little cherry virus-2 (LChV-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (ORFs). The sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of LChV-2 (variant USA6a). Both LChV-2/USA6a and LChV-2/USA6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. LChV-2/USA6b is more closely related to the partially determined genomic sequence of a Canadian isolate of LChV-2, strain LC5 (92.9% identity). LChV-2/USA6b has an unusual genomic organization compared to other members of the Closteroviridae. The LChV-2/USA6b genome is potentially ambi-sense, with a negative sense ORF0 at the 5' terminus, from which an 18.1 kDa protein of unknown function can be expressed in vitro. The N-terminal region of the LChV-2/USA6 ORF1a translation product does not code for a papain-like protease motif. ORF1 codes for a novel motif, of unknown function, also present in isolates of the Grapevine leafroll associated virus-3, (genus Ampelovirus) as well as viruses of the family Flexiviridae. ORF3 lacks an AUG start codon, but could potentially be expressed via read-through of the ORF2 stop codon. At the 3' end, there is a re-organization of encoded genes compared with other members of the Closteroviridae including separation of the coat protein and coat protein duplicate genes by 4 other genes as found for LChV-2/LC5.

Amino Acid Sequence↗
PubMed2005

Nucleotide sequence of Blackberry yellow vein associated virus, a novel member of the Closteroviridae.

The complete nucleotide sequence of a novel member of the genus Crinivirus (family Closteroviridae), isolated from blackberry and tentatively named Blackberry yellow vein associated virus, was determined. The virus possesses a bipartite genome. RNA 1 is 7,801 nucleotides in length and papain-like protease, methyltransferase, RNA helicase and RNA-dependent RNA polymerase motifs have been identified in the proteins coded for by this molecule. The polymerase is probably expressed via a +1 ribosomal frameshift, a common feature among members of the Closteroviridae. RNA 2 is 7,917 nucleotides long and encodes nine open reading frames, similar in size and position to orthologous genes of other criniviruses with the exception of a second hydrophobic peptide found near the 5' terminus of the molecule. Phylogenetic analysis revealed a close relationship between Blackberry yellow vein associated virus and Beet pseudo yellows virus, another crinivirus that infects small fruit crops.

Amino Acid Motifs↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed2002

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.

Amino Acid Sequence↗
PubMed2006

Comparative and functional genomics of closteroviruses.

The largest extant RNA genomes are found in two diverse families of positive-strand RNA viruses, the animal Coronaviridae and the plant Closteroviridae. Comparative analysis of the viruses from the latter family reveals three levels of gene conservation. The most conserved gene module defines RNA replication and is shared with plant and animal viruses in the alphavirus-like superfamily. A module of five genes that function in particle assembly and transport is a hallmark of the family Closteroviridae and was likely present in the ancestor of all three closterovirus genera. This module includes a homologue of Hsp70 molecular chaperones and three diverged copies of the capsid protein gene. The remaining genes show dramatic variation in their numbers, functions, and origins among closteroviruses within and between the genera. Proteins encoded by these genes include suppressors of RNA silencing, RNAse III, papain-like proteases, the AlkB domain implicated in RNA repair, Zn-ribbon-containing protein, and a variety of proteins with no detectable homologues in the current databases. The evolutionary processes that have shaped the complex and fluid genomes of the large RNA viruses might be similar to those that have been involved in evolution of genomic complexity in other divisions of life.

Amino Acid Sequence↗
PubMed2000

Closterovirus encoded HSP70 homolog and p61 in addition to both coat proteins function in efficient virion assembly.

Assembly of the viral genome into virions is a critical process of the virus life cycle often defining the ability of the virus to move within the plant and to be transmitted horizontally to other plants. Closteroviridae virions are polar helical rods assembled primarily by a major coat protein, but with a related minor coat protein at one end. The Closteroviridae is the only virus family that encodes a protein with similarity to cellular chaperones, a 70-kDa heat-shock protein homolog (HSP70h). We examined the involvement of gene products of Citrus tristeza virus (CTV) in virion formation and found that the chaperone-like protein plus the p61 and both coat proteins were required for efficient virion assembly. Competency of virion assembly of different CTV mutants was assayed by their ability to be serially passaged in Nicotiana benthamiana protoplasts using crude sap as inoculum, and complete and partial virus particles were analyzed by serologically specific electron microscopy. Deletion mutagenesis revealed that p33, p6, p18, p13, p20, and p23 genes were not needed for virion formation. However, deletion of either minor- or major-coat protein resulted in formation of short particles which failed to be serially transferred in protoplasts, suggesting that both coat proteins are required for efficient virion assembly. Deletion or mutation of HSP70h and/or p61 dramatically reduced passage and formation of full-length virions. Frameshift mutations suggested that the HSP70h and p61 proteins, not the RNA sequences, were needed for virion assembly. Substitution of the key amino acid residues in the ATPase domain of HSP70h, Asp(7) to Lys or Glu(180) to Arg, reduced assembly, suggesting that the chaperone-like ATPase activity is involved in assembly. Both HSP70h and p61 proteins appeared to contribute equally to assembly, consistent with coordinate functions of these proteins in closterovirus virion formation. The requirement of two accessory proteins in addition to both coat proteins for efficient assembly is uniquely complex for helical virions.

Amino Acid Substitution↗
PubMed2005

Complete nucleotide sequence of the RNA2 of the crinivirus tomato chlorosis virus.

The complete sequence of genomic RNA2 of Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae), isolate AT80/99 from Spain, was determined and compared with those from the other members of the genus sequenced to date. RNA2 is 8244 nucleotides (nt) long and putatively encodes nine ORFs that encompass the hallmark gene array of the family Closteroviridae, which includes a heat shock protein 70 family homologue, a 59 kDa protein, the coat protein, and a diverged coat protein. Phylogenetic analysis confirmed assignment of ToCV in the genus Crinivirus, being most similar to sweet potato chlorotic stunt virus and cucurbit yellow stunting disorder virus.

Base Sequence↗
PubMed2003

Nucleotide sequence and genome organization of Cucumber yellows virus, a member of the genus Crinivirus.

The genome of Cucumber yellows virus (CuYV), isolated in Japan from cucumber (Cucumis sativus L.), was completely sequenced and shown to be bipartite. CuYV RNA1 consisted of 7889 nucleotides and encompassed seven open reading frames (ORFs), which is typical of the Closteroviridae, including a heat-shock protein 70 homologue, a coat protein and a diverged coat protein (CPd). CuYV RNA2 consisted of 7607 nucleotides and included two ORFs: ORF1a potentially encoded a polyprotein containing putative papain-like protease, methyltransferase and helicase domains, and ORF 1b potentially encoded an RNA-dependent RNA polymerase, which is probably expressed via a +1 ribosomal frameshift. The size and organization of the CuYV genome are similar to those of Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, indicating that CuYV is a member of that genus, although CuYV differed in several points from LIYV.

Base Sequence↗
PubMed2004

Analysis of the RNA of Potato yellow vein virus: evidence for a tripartite genome and conserved 3'-terminal structures among members of the genus Crinivirus.

Double-stranded RNA preparations produced from potato plants graft-inoculated with a Peruvian isolate of Potato yellow vein virus (PYVV; genus Crinivirus, family Closteroviridae) contain five RNA species denoted RNA 1, RNA 2, RNA 3, x and y of approximately 8, 5.3, 3.8, 2.0 and 1.8 kbp, respectively. The complete nucleotide sequences of PYVV RNAs 1, 2 and 3 and Northern hybridization analysis showed that PYVV RNA 1 contained the replication module and an additional open reading frame (p7), while two distinct species, RNAs 2 and 3, contain the Closteroviridae hallmark gene array. Pairwise comparisons and phylogeny of genome-encoded proteins showed that PYVV shares significant homology with other criniviruses but is most closely related to the Trialeurodes vaporariorum-vectored Cucumber yellows virus. Secondary structure prediction of the 3'-untranslated regions of all three PYVV RNAs revealed four conserved stem-loop structures and a 3'-terminal pseudoknot structure, also predicted for all fully characterized members of the genus Crinivirus and some members of the genera Closterovirus and Ampelovirus.

3' Untranslated Regions↗
PubMed2006

Molecular analysis of double-stranded RNAs reveals complex infection of grapevines with multiple viruses.

The table grape variety "Waltham Cross" was infected with Leafroll and Shiraz Disease. To reveal specific viruses that are associated with the diseased plants, we used an RT-PCR-based strategy to determine partial genome sequences of these viruses. Upon cloning and sequencing of the RT-PCR products, we detected seven groups of viral variants that are related to four species of the Closteroviridae: Grapevine leafroll-associated virus 1, 2, 3, and 5, in addition to Rupestris stem pitting-associated virus. The population composition of GLRaV-2 and GLRaV-3-like viruses is complex and consists of two or three distinct groups of viral variants. Based on the consensus sequence of several GLRaV-2 strains, we designed a pair of broad-spectrum primers (GLR2-4 and GLR2-5) and used them to detect a range of GLRaV-2 variants from "Waltham Cross". Moreover, we identified a novel group of viral variants from the diseased grapevines, which possess a stretch of 19 nucleotides inserted in the 3' non-coding region as compared to strain "PN" and "93/955" for which the complete genomes have been sequenced. In contrast, the population composition of GLRaV-1 and GLRaV-5-like virus seems to be more uniform and each consists of a single viral variant. Furthermore, the central 5.7kb genomic region encompassing ORF1b-ORF4 of the GLRaV-1 isolate detected in "Waltham Cross" was sequenced. The new isolate is designated GLRaV-1 "WC", which differs from GLRaV-1 "Type" by 16% in nucleotide sequence. The taxonomic standing of the GLRaV-5-like and GLRaV-3-like viruses detected in "Waltham Cross" is discussed.

Biodiversity↗
PubMed2004

Complete nucleotide sequence and genome organization of Grapevine leafroll-associated virus 3, type member of the genus Ampelovirus.

This study reports on the complete genome sequence of Grapevine leafroll-associated virus 3, the type member of the genus Ampelovirus. The genome is 17 919 nt in size and contains 13 open reading frames (ORFs). Previously, the sequence of 13 154 nt of the 3'-terminal of the genome was reported. The newly sequenced portion contains a 158 nt 5' UTR, a single papain-like protease and a methyltransferase-like (MT) domain. ORF1a encodes a large polypeptide with a molecular mass of 245 kDa. With a predicted +1 frameshift, the large fusion protein generated from ORF1a/1b would produce a 306 kDa polypeptide. Phylogenetic analysis using MT domains further supports the creation of the genus Ampelovirus for mealy-bug-transmitted viruses in the family Closteroviridae.

Amino Acid Sequence↗
PubMed2003

Effects of modification of the transcription initiation site context on citrus tristeza virus subgenomic RNA synthesis.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a positive-sense RNA genome of about 20 kb organized into 12 open reading frames (ORFs). The last 10 ORFs are expressed through 3'-coterminal subgenomic RNAs (sgRNAs) regulated in both amounts and timing. Additionally, relatively large amounts of complementary sgRNAs are produced. We have been unable to determine whether these sgRNAs are produced by internal promotion from the full-length template minus strand or by transcription from the minus-stranded sgRNAs. Understanding the regulation of 10 sgRNAs is a conceptual challenge. In analyzing commonalities of a replicase complex in producing so many sgRNAs, we examined initiating nucleotides of the sgRNAs. We mapped the 5' termini of intermediate- (CP and p13) and low- (p18) produced sgRNAs that, like the two highly abundant sgRNAs (p20 and p23) previously mapped, all initiate with an adenylate. We then examined modifications of the initiation site, which has been shown to be useful in defining mechanisms of sgRNA synthesis. Surprisingly, mutation of the initiating nucleotide of the CTV sgRNAs did not prevent sgRNA accumulation. Based on our results, the CTV replication complex appears to initiate sgRNA synthesis with purines, preferably with adenylates, and is able to initiate synthesis using a nucleotide a few positions 5' or 3' of the native initiation nucleotide. Furthermore, the context of the initiation site appears to be a regulatory mechanism for levels of sgRNA production. These data do not support either of the established mechanisms for synthesis of sgRNAs, suggesting that CTV sgRNA production utilizes a different mechanism.

Base Sequence↗
PubMed2000

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.

Capsid↗
PubMed1999

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.

Animals↗
PubMed2000

The 23-kDa protein coded by the 3'-terminal gene of citrus tristeza virus is an RNA-binding protein.

The 23-kDa protein (p23), encoded by the 3'-proximal gene of the RNA of Citrus tristeza virus (CTV), was overexpressed in Escherichia coli fused to the maltose-binding protein and purified by affinity chromatography. Gel retardation and UV crosslinking assays demonstrated that p23 has the ability to cooperatively bind single-stranded RNA in a non-sequence-specific manner. Formation of the p23-RNA complex was dependent on the conformational state of p23 and on the presence of a basic region, but the complex was stable at high salt concentrations, suggesting that interactions other than those between the negatively charged RNA and the basic region of p23 are involved. Competition assays showed that the affinity of p23 for single-stranded and double-stranded RNA was similar but considerably higher than for single-stranded and double-stranded DNA. By use of a series of artificially generated mutants, the RNA-binding domain of p23 was mapped between positions 50-86, a region containing several basic amino acids and a putative zinc-finger domain. Additional p23-derivatives lacking the conserved residues presumably involved in coordinating the zinc ion showed RNA-binding activity, but with an apparent dissociation constant higher than the wild-type protein. These conserved residues might confer binding specificity or increase binding stability in vivo. Within the Closteroviridae family, p23 is the only protein characterized so far showing RNA-binding activity.

Chromosome Mapping↗
PubMed2001

Characterization of the cis-acting elements controlling subgenomic mRNAs of citrus tristeza virus: production of positive- and negative-stranded 3'-terminal and positive-stranded 5'-terminal RNAs.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has an approximately 20-kb positive-sense RNA genome with two 5' ORFs translated from the genomic RNA and 10 3' genes expressed via nine or ten 3'-terminal subgenomic (sg) RNAs. The expression of the 3' genes appears to have properties intermediate between the smaller viruses of the "alphavirus supergroup" and the larger viruses of the Coronaviridae. The sgRNAs are contiguous with the genome, without a common 5' leader, and are associated with large amounts of complementary sgRNAs. Production of the different sgRNAs is regulated temporally and quantitatively, with the highly expressed genes having noncoding regions (NCR) 5' of the ORFs. The cis-acting elements that control the highly expressed major coat protein (CP) gene and the intermediately expressed minor coat protein (CPm) gene were mapped and compared. Mutational analysis showed that the CP sgRNA controller element mapped within nts -47 to -5 upstream of the transcription start site, entirely within the NCR, while the CPm control region mapped within a 57 nt sequence within the upstream ORF. Although both regions were predicted to fold into two stem-loop structures, mutagenesis suggested that primary structure might be more important than the secondary structure. Because each controller element produced large amounts of 3'-terminal positive- and negative-stranded sgRNAs, we could not differentiate whether the cis-acting element functioned as a promoter or terminator, or both. Reversal of the control element unexpectedly produced large amounts of a negative-stranded sgRNA apparently by termination of negative-stranded genomic RNA synthesis. Further examination of controller elements in their native orientation showed normal production of abundant amounts of positive-stranded sgRNAs extending to near the 5'-terminus, corresponding to termination at each controller element. Thus, each controller element produced three sgRNAs, a 5'-terminal positive strand and both positive- and negative-stranded 3'-terminal RNAs. Therefore, theoretically CTV could produce 30-33 species of RNAs in infected cells.

Base Sequence↗
PubMed2002

Mutational analysis of the replication signals in the 3'-nontranslated region of citrus tristeza virus.

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb messenger-sense RNA genome consisting of 12 open reading frames with nontranslated regions (NTR) at the 5' and 3' termini. The 273 nucleotide (nt) 3'-NTR is highly conserved ( approximately 95%) among the sequenced CTV isolates in contrast to the highly diverse 5'-NTR sequences. The 3' replication signals were mapped to the 3' 234 nts within the NTR. This region of CTV does not contain a poly-A tract nor does it appear to fold as a tRNA-mimic. Instead, a computer-predicted thermodynamically stable secondary structure comprised of 10 stem-and-loop (SL) structures, referred to as SL1 to SL10 (5' to 3'), was common to all CTV isolates. This putative structure was used as a guide to examine the 3' requirements for replication in vivo. The resulting data suggest that a complex 3' structure is required for those functions that provide for efficient replication of CTV in vivo such as minus-strand initiation, regulation of strand asymmetry, effective translation of the myriad of viral mRNAs, or stability of RNAs. Deletions into the 3'-NTR, up to 66 nts from the 5' direction and 11 nts from the 3' direction, deleting or disrupting putative SL1, SL2 and SL3, or SL10, resulted in continued replication, suggesting that these sequences are not essential for basal-level replication, but are required for efficient replication. Predicted stem loops 3 through 10 were examined by mutations designed to alter the primary structures while preserving the secondary structures. Mutations designed to disrupt the predicted stems of SL3, SL5, SL7, SL9, or SL10 resulted in substantially reduced levels of replication, while compensatory mutations resulted in partial restorations of replication, suggesting that these predicted secondary structures are involved in replication. Also, the putative loop sequences of SL5, SL6, SL7, and SL9 tolerated mutagenesis with continued but reduced levels of replication. In contrast, all mutations introduced into putative SL4, SL8, and the stem of SL6 prevented replication, suggesting that the primary structure of these regions make up the core of the 3' replication signal. The 3' triplet, CCA, was shown to be necessary for efficient replication, but deletion of eleven nts to expose an internal CCA resulted in continued replication.

3' Untranslated Regions↗