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At least 19 recordsLinked to original sources

PubMed1998

Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member.

The entire genome of grapevine leafroll-associated closterovirus-2 (GLRaV-2), except the exact 5' terminus, was cloned and sequenced. The sequence encompasses nine open reading frames (ORFs) which include, in the 5' to 3' direction, an incomplete ORF1a encoding a putative viral polyprotein and eight ORFs that encode proteins of 52 kDa (ORF1b), 6 kDa (ORF2), 65 kDa (ORF3), 63 kDa (ORF4), 25 kDa (ORF5), 22 kDa (ORF6), 19 kDa (ORF7) and 24 kDa (ORF8) respectively, and 216 nucleotides of the 3' untranslated region. An incomplete ORF1a potentially encoded a large polyprotein containing the conserved domains characteristic of a papain-like protease, methyltransferase and helicase. ORF1b potentially encoded a putative RNA-dependent RNA polymerase. The expression of ORF1b may be via a +1 ribosomal frameshift mechanism, similar to other closteroviruses. A unique gene array, which is conserved in other closteroviruses, was also identified in GLRaV-2; it includes genes encoding a 6 kDa small hydrophobic protein, 65 kDa heat shock protein 70, 63 kDa protein of function unknown, 25 kDa coat protein duplicate and 22 kDa coat protein. Identification of ORF6 (22 kDa) as the coat protein gene was further confirmed by in vivo expression in E. coli and immunoblotting. Phylogenetic analysis comparing different genes of GLRaV-2 with those of other closteroviruses demonstrated a close relationship with beet yellows virus (BYV), beet yellow stunt virus and citrus tristeza virus. GLRaV-2 is the only closterovirus, so far, that matches the genome organization of the type member of the group, BYV, and thus can be unambiguously classified as a definitive member of the genus Closterovirus.

Amino Acid Sequence↗

PubMed1998

Nucleotide sequence of the 3'-terminal two-thirds of the grapevine leafroll-associated virus-3 genome reveals a typical monopartite closterovirus.

The RNA genome of grapevine leafroll-associated closterovirus-3 (GLRaV-3) was cloned as a cDNA generated from GLRaV-3-specific dsRNA, and a partial genome sequence of 13154 nucleotides (nt) including the 3' terminus was determined. The sequenced portion contained 13 open reading frames (ORFs) potentially encoding, in the 5'-3' direction, proteins of > 77 kDa (ORF1a; helicase, HEL), 61 kDa (ORF1b; RNA-dependent RNA polymerase, RdRp), 6 kDa (ORF2), 5 kDa (ORF3, small transmembrane protein), 59 kDa (ORF4; heat shock protein 70, HSP70), 55 kDa (ORF5), 35 kDa (ORF6; coat protein, CP), 53 kDa (ORF7; diverged coat protein, CPd), 21 kDa (ORF8), 20 kDa (ORF9), 20 kDa (ORF10), 4 kDa (ORF11), 7 kDa (ORF12), and an untranslated region of 277 nt. ORF1b is probably expressed via a +1 ribosomal frameshift mechanism, most similar to that of lettuce infectious yellows virus (LIYV). Phylogenetic analysis using various gene sequences (HEL, RdRp, HSP70 and CP) clearly demonstrated that GLRaV-3, a mealybug-transmissible closterovirus, is positioned independently from aphid-transmissible monopartite closteroviruses (beet yellows, citrus tristeza and beet yellows stunt) and whitefly-transmissible bipartite closterovirus (lettuce infectious yellows, LIYV). However, another alleged mealybug-transmissible closterovirus, little cherry virus, was shown to be more closely related to the whitefly-transmissible LIYV than to GLRaV-3.

Amino Acid Sequence↗

PubMed1995

Genome structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-transmitted, bipartite closterovirus.

We report the complete nucleotide sequences of lettuce infectious yellows virus (LIYV) RNAs 1 and 2. LIYV RNA 1 is 8118 nucleotides and includes three open reading frames (ORFs). Computer-assisted analysis of LIYV RNA 1 ORFs identified domains for a papain-like protease, methyltransferase (MTR), RNA helicase (HEL), and RNA-dependent RNA polymerase (RdRp). We suggest that the RdRp domain is expressed independently of the other replication-associated domains via a + 1 ribosomal frameshift. Amino acid sequences of the MTR, HEL, and RdRp show highly significant similarity to the homologous sequences from other closteroviruses and lower similarity to the respective proteins of tobamoviruses, tobraviruses, hordeiviruses, bromoviruses, and furoviruses. LIYV RNA 2 is 7193 nucleotides and includes six ORFs. These ORFs include a gene array that is characteristic of the closteroviruses: ORFs encoding a small membrane protein, a homologue of the HSP70 family of chaperone proteins, a protein whose function is unknown, the coat protein, and a diverged duplicate of the coat protein. LIYV is distinguished from the monopartite closteroviruses in the following ways: its genome consists of two RNAs, the positions of the coat protein gene and its diverged duplicate are reversed, and LIYV includes ORFs that are unrelated to ORFs found in other closteroviruses.

Amino Acid Sequence↗

PubMed1996

Organization of the 3'-terminal half of beet yellow stunt virus genome and implications for the evolution of closteroviruses.

The 3'-terminal half of the beet yellow stunt virus (BYSV) genome 10,545 nt, has been cloned and sequenced. The sequenced portion of the BYSV genome encompasses 10 open reading frames (ORFs) and 241 nt of the 3' untranslated region. The sequence spans, in the 5' to 3' direction, the C-terminal region of the replication-associated polyprotein gene (ORF 1a) which includes the set of motifs typical of helicases (HEL), the entire 53-kDa polymerase (RdRp) gene (ORF 1b), and genes encoding 30-kDa (ORF 2), 6-kDa (ORF 3), 66-kDa (ORF 4), 61-kDa (ORF 5), 25-kDa (ORF 6), 23.7-kDa (coat protein, CP) (ORF 7), 18-kDa (ORF 8), and 22-kDa (ORF 9) proteins. The double-stranded RNA "replicative form" of the BYSV was demonstrated to have a nontemplate G residue at the 3' terminus of the (+) strand. The RdRp of BYSV is presumably expressed via a +1 ribosomal frameshift. The five-gene module conserved among closteroviruses was identified in BYSV; it includes a gene array coding for a 6-kDa small hydrophobic protein, a 66-kDa homolog of the cellular HSP70 heat shock proteins, a 61-kDa protein, and a 25-kDa diverged copy of the CP followed by the CP gene itself. Phylogenetic analysis of the replication-associated HEL and RdRp domains as well as proteins from the five-gene module demonstrated the closest relationship between BYSV and two other closteroviruses, beet yellows (BYV) and citrus tristeza (CTV) viruses. Like CTV, the BYSV genome contains a 30-kDa protein gene between the RdRp and the 6-kDa protein genes, and like BYV it has only two genes downstream of the CP gene. The organization of the BYSV genome appears to be intermediate between BYV and CTV, which suggests that these three viruses might represent three distinct but probably close stages in the closterovirus evolution.

Amino Acid Sequence↗

PubMed1996

Beet pseudo-yellows virus is an authentic closterovirus.

We have previously used RT-PCR to amplify a 251 base pair fragment diagnostic for beet pseudo-yellows virus (BPYV) infection from several BPYV infected samples from Europe and the USA. This has now been shown by sequencing to represent a fragment of a heat shock protein (HSP)70 gene. Sequence analysis of the fragment revealed that it is closely similar to the C-terminal sequence of the HSP70 homologue gene of lettuce infectious yellows virus, a definitive closterovirus, and also has obvious sequence similarities to the HSP70 genes of the other previously sequenced closteroviruses citrus tristeza virus and beet yellows virus. This observation confirms that BPYV is an authentic closterovirus with a wide geographical distribution.

Amino Acid Sequence↗

PubMed1997

Complete genome structure and phylogenetic analysis of little cherry virus, a mealybug-transmissible closterovirus.

The 5'-terminal genomic region (8597 nt) of little cherry virus (LChV), a mealybug-borne closterovirus, was cloned from double-stranded RNA, and its sequence determined to complete the 16934 nt sequence of the monopartite LChV RNA genome. In the 5' to 3' direction, the sequence encompasses ORF 1a, encoding the conserved replicative domains of methyltransferase and helicase, and ORF 1b, encoding RNA polymerase. ORFs 1a and 1b partially overlap (in O/+1 configuration), and the LChV replicase is probably expressed by ribosomal frameshifting as a fusion product with a molecular mass of 318 kDa. The N-terminal part of the ORF 1a product contains a papain-like cysteine proteinase (PCP) domain with a predicted cleavage site between Gly-619 and Ser-620. The PCP and the upstream protein domains can be aligned with the equivalent parts of the leader proteins encoded by the whitefly-transmitted lettuce infectious yellows and sweet potato sunken vein closteroviruses. Phylogenetic reconstruction based on the aligned RNA polymerase sequences clearly suggests that the aphid-transmissible and whitefly-transmissible closteroviruses represent two distinct evolutionary lineages, with the mealybug-transmissible LChV being the most remote member of the 'whitefly' lineage.

Amino Acid Sequence↗

PubMed1998

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.

Closterovirus↗

PubMed2000

Genetic Diversity and Evolution of Closteroviruses.

The family Closteroviridae comprises more than 30 plant viruses with flexuous, filamentous virions and includes representatives with either mono- or bipartite positive-strand ssRNA genomes. Closteroviruses are transmitted semipersistently by insects from three families of Homoptera, in infected plants are associated with phloem tissue, and demonstrate an astonishing genetic diversity that suggests extensive, on-going evolution. Phylogenetic analyses of their replicative genes as well as the conserved HSP70 demonstrate that closteroviruses co-evolved with their insect vectors, resulting in three major lineages, i.e. aphid-, mealybug-, and whitefly-transmitted viruses. Closteroviruses apparently represent an ancient and diverse virus family that may pose threats to agriculture and needs serious attention.

Journal Article↗

PubMed1994

Nucleotide sequence and organization of eight 3' open reading frames of the citrus tristeza closterovirus genome.

The citrus tristeza closterovirus (CTV) RNA genome was cloned as cDNA generated from both CTV-specific double-stranded RNA and genomic RNA, and the sequence of the 3' 7292 nucleotides was determined. The sequenced portion contained eight open reading frames potentially encoding, in the 5' to 3' direction, proteins with the apparent molecular weights of 65, 61, 27, 25 (capsid protein, CP), 18, 13, 20, and 23 kDa, and a potential noncoding region of 277 nucleotides. The 65-kDa protein is a viral homolog of cellular hsp70 heat shock proteins (hsp), the 61-kDa protein is distantly related to the hsp90 proteins, and the 27-kDa protein is a diverged copy of the CP. Database searches did not identify any protein sequences of significant similarity to the remaining four ORFs downstream of the CP. A specific four-gene module consisting of the hsp70 protein, the hsp90-related protein, the diverged copy of the CP, and the CP itself was found to be common in organization between CTV and beet yellows closterovirus. All four proteins in this module were highly conserved, indicating that these viruses probably have evolved from a common ancestor.

Amino Acid Sequence↗

PubMed1996

In vitro transcripts from cloned cDNAs of the lettuce infectious yellows closterovirus bipartite genomic RNAs are competent for replication in Nicotiana benthamiana protoplasts.

Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived capped in vitro transcripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned cDNAs of a closterovirus genome.

Capsid↗

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↗

PubMed1997

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants.

A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the 3' end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated M(r) of 34866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other closteroviruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot.

Agrobacterium tumefaciens↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed1994

Screening of the closterovirus genome by degenerate primer-mediated polymerase chain reaction.

The genome of beet yellows virus (BYV), the type representative of the closterovirus group, encodes a homologue of the cellular heat-shock protein (HSP) 70 family. A pair of degenerate primers targeted to motifs A and E, which are highly conserved in HSP70s, was synthesized. Genomes of several definite and possible members of the closterovirus group were screened for the presence of the HSP70 gene with PCR using these degenerate primers. BYV, citrus tristeza virus (CTV), beet yellow stunt virus (BYSV) and carnation necrotic fleck virus templates produced 1 kb amplification products, which were shown by sequencing to represent fragments of the respective HSP70 genes. Further screening was performed with an additional degenerate primer targeted to the motif IV of the putative viral polymerase. This degenerate primer and specific primers complementary to the 5' region of the HSP70 genes of the respective viruses were used to estimate the distance between polymerase motif IV and the start point of the HSP70 gene for BYV (approximately 1.1 kb), CTV and BYSV (around 2.0 kb) by PCR. The amplified genome regions of CTV (3026 nucleotides) and BYSV (2837 nucleotides) were cloned and sequenced. CTV and BYSV were found to encode the gene for an additional 30K (BYSV) or 33K (CTV) protein between the polymerase and the small hydrophobic protein genes, which was absent in BYV. These two 30K proteins displayed very weak similarity to each other, unlike the highly conserved polymerases, hydrophobic proteins and HSP70s of BYV, CTV and BYSV. Degenerate primer-mediated PCR proved to be an efficient tool for rapid screening and subsequent cloning of the viral genomes.

Base Sequence↗

PubMed1998

Beet yellows closterovirus HSP70-like protein mediates the cell-to-cell movement of a potexvirus transport-deficient mutant and a hordeivirus-based chimeric virus.

It has been suggested that the beet yellows closterovirus (BYV)-encoded p65 protein, a homologue of HSP70 cell chaperones, plays a role as a virus movement protein (MP). To test this hypothesis, we used two types of complementation experiments with plant viruses containing the triple gene block (TGB) of MP genes. In one, the BYV p65 gene was cloned into a 35S promoter plasmid and introduced into Nicotiana benthamiana plants by microprojectile bombardment along with the 35S promoter-driven GUS gene-tagged cDNA of a transport-deficient potexvirus mutant. Transient expression of p65 complemented the mutant as visualized by the significant increase in the number of cells expressing the GUS reporter gene in the infection foci. In the other test, the p65 gene was inserted into the infectious cDNA of the hordeivirus RNA beta component to replace either the 58 kDa MP gene or the whole TGB. Inoculation of Chenopodium quinoa and Chenopodium amaranticolor plants with the T7 transcripts of the chimeric RNA beta, together with the hordeivirus RNA alpha and RNA gamma, caused symptomless infection in inoculated leaves detected by hybridization of the total leaf RNA with a specific cDNA probe. The ability of BYV p65 to substitute for the potexvirus or hordeivirus MPs provides direct evidence for its involvement in the cell-to-cell movement of closterovirus infection.

Chimera↗

PubMed1999

The minor coat protein of beet yellows closterovirus encapsidates the 5' terminus of RNA in virions.

Filamentous particles of beet yellows closterovirus (BYV) are built of two related capsid proteins, of which the minor species, p24, forms a 75 nm tail at one end of the virion. In the present work, we used polyclonal antibodies against p24 for isolating the 'tailed' virion segments from sonicated BYV particle preparations. The [gamma-32P]ATP-labelled RNA obtained from the antibody-selected particle segments consistently showed stronger hybridization with the 5'-terminal BYV cDNA clones than with the 3'-terminal cDNA clones. These data clearly indicate that it is the 5'-terminal portion of the closterovirus RNA genome that is encapsidated by p24.

Capsid↗

PubMed2003

Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus.

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

Aizoaceae↗

PubMed1997

Transcriptional strategy of closteroviruses: mapping the 5' termini of the citrus tristeza virus subgenomic RNAs.

Citrus tristeza virus (CTV) induces formation of a nested set of at least nine 3' coterminal subgenomic RNAs (sgRNAs) in infected tissue. The organization and expression of the 19,296-nucleotide (nt) CTV genome resembles that of coronaviruses, with polyprotein processing, translational frameshifting, and multiple sgRNA formation, but phylogenetically the CTV polymerase, like polymerases of other closteroviruses, belongs to the Sindbis virus-like lineage of RNA virus polymerases. Both positive-strand RNA virus supergroups, coronaviruses and Sindbis-like viruses, utilize different mechanisms of transcription. To address the mechanism of CTV transcription, 5' termini for the two most abundant sgRNAs, 1.5 and 0.9 kb, respectively, were mapped by runoff reverse transcription. The two sgRNAs were demonstrated to have 48- and 38-nt 5' untranslated regions (5'-UTRs), respectively. The 5'-UTR for the 1.5-kb RNA was cloned, sequenced, and demonstrated to be colinear with the 48-nt genomic sequence upstream of the initiator codon of the respective open reading frame 10, i.e., to be of continuous template origin. The data obtained suggest that the sgRNA transcription of CTV is dissimilar from the coronavirus transcription and consistent with the transcriptional mechanism of other Sindbis-like viruses. Thus, the Sindbis virus-like mechanism of transcription of the positive-strand RNA genomes might be successfully utilized by the closterovirus genome of up to 19.3 kb with multiple sgRNAs.

Base Sequence↗