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PubMed2003

Occurrence and sequences of Lily mottle virus and Lily symptomless virus in plants grown from imported bulbs in Zhejiang province, China.

Degenerate primers were used to amplify virus sequences from imported lilies in Zhejiang province, China. Two viruses, Lily mottle virus (LMoV, genus Potyvirus) and Lily symptomless virus (LSV, genus Carlavirus) were detected, purified and completely sequenced from a mixed infection in a plant raised from bulbs imported from the Netherlands. The sequence of LMoV was 9644 nt long and encoded a polyprotein of 3095 amino acids with a calculated M(r) of 351.0 kDa that had only 45.1-54.4% identity to other completely sequenced potyviruses. Phylogenetic analysis of the complete polyproteins of members of the genus demonstrated that LMoV was distantly grouped with LYSV, BYMV and ClYVV. Two partial LMoV sequences from different cultivars were identical to one another and very similar (98.3% identical nucleotides) to the corresponding region of the complete sequence. Analysis of the coat protein sequences of LMoV isolates revealed two subgroups, corresponding to the earlier "Tulip breaking virus lily strain" and "Tulip band breaking virus" isolates. Our newly-determined isolates showed an extremely close relationship to the first of these. The LSV sequence was 8393 nucleotides long and had the typical carlavirus genome organization. The ORF1 protein was most closely related to that of Blueberry scorch virus (57.2% identical amino acids). Sequences of 1796 nt at the 3'-end of three additional LSV isolates from different cultivars were very similar (>98% identical nucleotides) to the corresponding region of the complete sequence. This is the first report of complete sequences for LMoV and LSV.

Amino Acid Sequence↗
PubMed2004

Complete nucleotide sequence of the RNA 1 of a grapevine isolate of Arabis mosaic virus.

The complete nucleotide sequence of the genomic RNA 1 of the grapevine isolate NW (Neustadt an der Weinstrasse) of Arabis mosaic virus (ArMV) was determined. It is 7334 nucleotides long excluding the poly(A) tail, and contains one long open reading frame encoding a polypeptide of 2284 amino acids. The 5' and 3' non-coding regions were 227 and 252 nucleotides long respectively, and showed stretches of high identity with the corresponding 5' and 3' non-coding regions of ArMV-NW RNA 2. The analysis of the amino acid sequence of the polyprotein encoded by the RNA 1 of the ArMV-NW showed that the conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cystein protease, and the RdRp core domains, were all present. Amino acid sequence comparisons between the polyproteins encoded by the RNAs 1 of ArMV-NW and other nepoviruses showed 75% identity with the GFLV-F13, and up to 36% with other nepoviruses.

Genome, Viral↗
PubMed2004

A new potato virus in a new lineage of picorna-like viruses.

On constructing a cDNA library for potato, 'contaminating' sequences with a significant identity to Apple latent spherical virus (ALSV) were found. Determination of the remaining genome sequence indicated the presence of a bipartite virus with an RNA1 and 2 of 7034 and 3315 nucleotides, respectively, excluding a poly(A)tail. RNA1 encodes a single polyprotein (233 kDa) and shares highest amino acid identity with ALSV at 65%. Conserved amino acid motifs typical for helicase, protease and RNA-dependent polymerase (RdRp) functions are present. RNA2 encodes a single polyprotein (106 kDa) with amino acid identities to the flat apple isolate of Cherry rasp leaf virus (CRLV-FA) (97%) and ALSV (70%), suggesting this is a potato strain of CRLV (CRLV-pot). Phylogenetic analysis using the RdRp region shows that this virus falls within a group separate from the Comoviridae that includes members of the Sequiviridae and the taxonomically unassigned viruses ALSV, Strawberry mottle virus, Satsuma dwarf virus and Navel orange infectious mottling virus. Other regions of the genome have highest identities with both plant and animal infecting members of the picorna-like virus superfamily. The evolutionary context of CRLV-pot and related viruses is discussed. Similar viral sequences from an EST library of peppermint are also analysed.

5' Untranslated Regions↗
PubMed2006

Konjak mosaic virus: the complete nucleotide sequence of the genomic RNA and its comparison with other potyviruses.

Konjak mosaic virus (KoMV) belongs to the genus Potyvirus, family Potyviridae. The complete nucleotide sequence of KoMV F isolate (KoMV F) was determined. The genome is 9,544 nucleotides long excluding the 3' terminal poly A tail and encodes a typical potyviral 350-kDa polyprotein of 3,087 amino acids. Phylogenetic analysis using known potyvirus polyproteins shows that KoMV constitutes a branch with yam mosaic virus, close to another branch including Japanese yam mosaic virus, turnip mosaic virus, scallion mosaic virus and lettuce mosaic virus. The 3' terminal 1,842 nucleotides of a different isolate of KoMV, K-2, was also determined, covering the C-terminal 292 amino acids of the nuclear inclusion protein b (NIb), coat protein (CP), and the 3' untranslated region. The amino acid sequences of the KoMV F CP and the nucleotide sequences of the KoMV F 3' untranslated region showed 92.5 and 90.5% identity to the corresponding genes of K-2, 88.7-96.8 and 92.7-94.4% to those of Zantedeschia mosaic virus (ZaMV) isolates, 87.5-89.7% and 85.5-90.3% to those of Japanese hornwort mosaic virus (JHMV) isolates. These results showed that KoMV is a distinct potyvirus and that KoMV, ZaMV, and JHMV are members of the same potyvirus species. Considering that KoMV was the first of these to be described, ZaMV and JHMV may be considered isolates of KoMV.

Amorphophallus↗
PubMed2006

The complete genome sequence of pepper severe mosaic virus and comparison with other potyviruses.

The complete nucleotide sequence of pepper severe mosaic virus (PepSMV) was determined. The viral genome consisted of 9890 nucleotides, excluding a poly (A) tract at the 3' end of the genome. The PepSMV RNA genome encoded a single polyprotein of 3085 amino acid residues, resulting in ten functionally distinct potyviral proteins. The lengths of the 5' nontranslated region (NTR) and the 3' NTR were 164 and 468 nucleotides, respectively. The genome organization of the virus was typical for members of the genus Potyvirus in the family Potyviridae. The coat protein amino acid sequence identity between PepSMV and the other 45 potyviruses ranged from 53.4 to 79.7%. Sequence alignments and phylogenetic analyses of the potyviral polyprotein sequences revealed that PepSMV was the closest to potato virus Y (PVY) and closely related to members of the PVY subgroup. Our genome sequence data clearly confirmed that PepSMV belongs to a separate species in the genus Potyvirus.

3' Untranslated Regions↗
PubMed2000

Complete nucleotide sequence of the genomic RNA of a mild strain of Japanese yam mosaic potyvirus.

We determined the complete nucleotide sequence of a mild strain of Japanese yam mosaic potyvirus (JYMV-M) and compared it with the published sequence of severe strain of JYMV (JYMV-J1). The genomic RNA of JYMV-M is 9,760 nucleotides (nts) in length, excluding the poly (A) tail, and encodes a polyprotein of 3,132 amino acids. Among nine potential cleavage sites, only the P1 and NIa recognition sites (between 6K1 and CI) had different sequences from those of JYMV-J1. The data confirm the strain status of these two viruses with 91.1% sequence identity for the polyprotein and approximately 94-97% identities for HC, CI, NIa, NIb and CP. The most divergent products P1 and P3 had 62% and 90% sequence identities respectively.

Base Sequence↗
PubMed1997

Divergence and conservation of the genomic RNAs of Taiwan and Hawaii strains of papaya ringspot potyvirus.

The complete nucleotide sequence of the genome of a Taiwan isolate of papaya ringspot potyvirus (PRSV YK) was determined from three overlapping cDNA clones and by direct RNA sequencing. Comparison was made with the reported Hawaii isolate of PRSV HA. Both genomes are 10,326 nucleotides long, excluding the poly(A)-tail. They encode a polyprotein of 3344 amino acids with a 5' leader of 85 nucleotides and a 3' non-translated region of 209 nucleotides. The two genomes share an overall nucleotide identity of 83.4% and an amino acid identity of 90.6%. The 3' non-translated regions show 92.3% identity. The first 23 nucleotides of the leaders are identical, while the remaining parts of the leaders only show 51.6% identity. The P1 protein genes of the two isolates are very different, with 70.9% nucleotide identity and 66.7% encoded amino acids identity. However, the other viral proteins of the two virus isolates are similar, with a 82.5-89.8% nucleotide identity of their genes and 91.2-97.6% amino acid identity, indicating that they are strains of the same potyvirus. Analysis of the ratios of nucleotide differences to the actual amino acid changes revealed that there are only 2.63 nucleotide changes for each amino acid change in the P1 protein, whereas for the other proteins 4.0-16.4 nucleotide changes are required for each amino acid replacement. The P1 protein has 58% of all the differences of polyprotein. The unusual variation in the leader sequences and the P1 proteins suggests that the two PRSV strains were derived from different evolutionary pathways in different geographic areas.

Amino Acid Sequence↗
PubMed1997

Expression and interaction of the hepatitis C virus structural proteins and the 5' untranslated region in baculovirus infected cells.

The structural proteins, core, E1 and a C-terminal truncated E2 (C-E1-E2p) as well as the 5'UTR linked to the core gene (5'UTR-C) of the hepatitis C virus were expressed in Sf9 cells. Expression of the C-E1-E2p polyprotein from a single vector resulted in expression of multiple forms suggesting that the cleavage of the polyprotein in the insect cells is incomplete. The structural proteins were expressed as insoluble forms and were solubilized by freeze/thaw cycles or detergent treatment. Analysis of the co-expressed (5'UTR-C and C-E1-E2p) proteins through a 20-60% sucrose gradient revealed that the core protein migrated rapidly into the gradient, in common with the sedimentation profile for core protein expressed from the core gene only. Neither E1 nor E2 proteins were involved in the core-core protein interaction. Expression of the 5'UTR-C in insect cells resulted in a high level of mRNA transcription but no translation of core protein, presumably due to a failure to initiate HCV translation by an internal ribosome entry mechanism, since the mRNA transcript was translated in vitro, suggesting that the 5'UTR can influence HCV tropism.

Animals↗
PubMed1998

Complete nucleotide sequence of wheat yellow mosaic bymovirus genomic RNAs.

The complete sequences of wheat yellow mosaic bymovirus (WYMV) RNA1 and RNA2 were determined. RNA1 is 7636 nucleotides long [excluding the 3'-poly(A)], and codes for a 269 kDa polyprotein of 2,404 amino acids which contains the capsid protein (CP) at the C terminus and seven putative nonstructural proteins. RNA2 is 3,659 nucleotides long and codes for a polyprotein of 904 amino acids which contains a 28 kDa putative proteinase and a 73 kDa polypeptide. These functional proteins are arranged as in RNA1 and RNA2 of barley yellow mosaic bymovirus (BaYMV). Comparisons with the sequence reported for the 3' half of RNA1 of wheat spindle streak mosaic bymovirus (WSSMV) from Southern France show that WYMV and WSSMV have a similar genetic organization. However, WYMV and WSSMV share only 77% amino acid sequence identity in their deduced CPs in spite of their close serological relationship, and 74% nucleotide sequence identity in their 3' non-coding regions. Thus, the sequence data indicate that WYMV and WSSMV are not strains of the same virus, which has long been suggested, but are distinct virus species within the genus Bymovirus of the family Potyviridae.

Amino Acid Sequence↗
PubMed1999

Complete nucleotide sequence of the genomic RNA of a Japanese yam mosaic virus, a new potyvirus in Japan.

We determined the complete nucleotide sequence of a potyvirus purified from a Japanese yam plant. The genomic RNA of this virus is 9,757 nucleotides (nts) in length, excluding the 3'-terminal poly(A) tail. It contains a single open reading frame (ORF) encoding a polyprotein of 3130 amino acids (aa) with a calculated Mr of 356,793. The genomic organization of this potyvirus is similar to that of other members of the genus Potyvirus and nine potential cleavage sites for the viral proteinase were found by comparison of its sequence with those available for other potyviruses. The nucleotide sequence and genome characteristics show that this isolate is a new potyvirus species. Its polyprotein differs substantially from Yam mosaic virus (YMV) (50% amino acid sequence identity) and fourteen other potyvirus species examined (44-59% identity). Although this potyvirus has been classified as YMV, our results suggest that the potyvirus infectious to the Japanese yam plant in Japan is distinct from YMV. Therefore, we propose that the Japanese yam potyvirus should be designated as Japanese yam mosaic virus (JYMV).

Amino Acid Sequence↗
PubMed1999

Identification of genes encoding for the cocksfoot mottle virus proteins.

Cocksfoot mottle sobemovirus (CfMV) has a monopartite single-stranded positive-sense RNA genome. In wheat germ extract, in vitro translation of CfMV virion RNA resulted in the production of four major proteins of 100, 71, 34 and 12 kDa. In this paper we show the individual genes from which these polypeptides are synthesised. Polyclonal antisera against the proteins encoded by all open reading frames (ORFs) of CfMV were produced. Antibodies were used to immunoprecipitate the in vitro translation products of CfMV genomic RNA as well as the coupled in vitro transcription/translation products of individual viral genes. We demonstrate that the 12, 71, and 100 kDa CfMV proteins are synthesised from the bicistronic genomic RNA of the virus. The CfMV 12 kDa protein is produced from ORF1, the 71 kDa protein from ORF2a (and does not result from the proteolytic cleavage of the polyprotein) and the 100 kDa protein is a polyprotein encoded by ORFs 2a and 2b by a -1 ribosomal frameshift. ORF2b alone does not direct any in vitro protein synthesis. CfMV 34 kDa protein is a coat protein synthesised from the virion-packed subgenomic RNA. The translational strategies of different sobemoviruses are also discussed in this paper.

Animals↗
PubMed2000

Nucleotide sequence and synthesis of infectious RNA from cloned cDNA of broad bean wilt virus 2 RNA 2*.

The complete nucleotide sequence of broad bean wilt virus -2 (BBWV-2) RNA 2 was determined. The sequence is 3593 nucleotides in length, and contains a single open reading frame which has the potential to encode a 119 kDa polyprotein. Direct amino acid sequencing of the virus coat proteins suggests that the polyprotein is processed to produce three proteins, 52 kDa, 44 kDa and 22 kDa in size. A comparison of several fabaviruses in terms of nucleotide sequence and amino acid sequences showed that BBWV-2 isolates display high sequence identity. A full-length cDNA constructed under the control of the T7 promoter could be successfully transcribed to produce biologically active RNA. This is the first report on generating infectious transcripts of BBWV-2 RNA 2.

Base Sequence↗
PubMed2000

Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion.

The complete nucleotide sequence of the genomic RNA of a vaccine strain (SP) of porcine reproductive and respiratory syndrome virus (PRRSV) was determined. It shares approximately 94% identity of nucleotide sequence with two recently reported North American strains, 16244B and VR2332, but only 78% with a European strain, Lelystad virus (LV). Its genome is the longest among the four published complete sequences of PRRSV, due to an insertion in the Nsp2-encoding region. Compared to Nsp2 of the North American strains and the European strain, the predicted Nsp2 of strain SP contains 36 and 155 amino acid insertions, respectively, near the C-terminus, in addition to several highly variable regions. The insertion shows no homology with any equivalent arterivirus proteins. This high sequence disparity of Nsp2 among different PRRSV isolates suggested that it could be used as a marker to differentiate PRRSV genotypes. The 5' RACE and primer extension analysis of three North American strains demonstrated that the utmost 5'-end nucleotides are conserved among PRRSV strains isolated from two continents. The predicted polyprotein 1 a/b contains conserved proteinase, polymerase and helicase domains responsible for polyprotein processing, RNA transcription and replication.

Animals↗
PubMed2000

Expression of the hepatitis E virus ORF1.

Hepatitis E virus (HEV) is an unclassified, plus-strand RNA virus whose genome contains three open reading frames (ORFs). ORF1, the 5' proximal ORF of HEV, encodes nonstructural proteins involved in RNA replication which share homology with the products of the corresponding ORF of members of the alphavirus-like superfamily of plus-strand RNA viruses. Among animal virus members of this superfamily (the alphavirus and rubivirus genera of the family Togaviridae), the product of this ORF is a nonstructural polyprotein (NSP) that is cleaved by a papain-like cysteine protease (PCP) within the NSP. To determine if the NSP of HEV is similarly processed, ORF1 was introduced into a plasmid vector which allowed for expression both in vitro using a coupled transcription/translation system and in vivo using a vaccinia virus-driven transient expression system. A recombinant vaccinia virus expressing ORF1 was also constructed. Both in vitro and in vivo expression under standard conditions yielded only the full-length 185 kDa polyprotein. Addition of co-factors in vitro, such as divalent cations and microsomes which have been shown to activate other viral proteases, failed to change this expression pattern. However, in vivo following extended incubations (24-36 hours), two potential processing products of 107 kDa and 78 kDa were observed. N- and C-terminus-specific immunoprecipitation and deletion mutagenesis were used to determine that the order of these products within the NSP is NH2-78 kDa-107 kDa-COOH. However, site-specific mutagenesis of Cys483, predicted by computer alignment to be one member of the catalytic dyad of a PCP within the NSP, failed to abolish this cleavage. Additionally, sequence alignment across HEV strains revealed that the other member of the proposed catalytic dyad of this PCP, His590, was not conserved. Thus, the cleavage of the NSP observed following prolonged in vivo expression was not mediated by this protease and it is doubtful that a functional PCP exists within the NSP. Attempts to detect NSP expression and processing in HEV-infected primary monkey hepatocytes were not successful and therefore this proteolytic cleavage could not be authenticated. Overall, the results of this study indicate that either the HEV NSP is not processed or that it is cleaved at one site by a virally-encoded protease novel among alpha-like superfamily viruses or a cellular protease.

Amino Acid Sequence↗
PubMed2001

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus.

The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5' non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of M(r) 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326-337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E325-T326. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E445-T446 based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a--1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via--1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters.

Base Sequence↗
PubMed2005

Expression of a self-processing, pathogen resistance-enhancing gene construct in Arabidopsis.

A gene cassette, p35S-CNO, was designed to express three gene products driven by a single constitutive CaMV 35S promoter. The individual coding regions were linked in frame to produce a single polyprotein, using spacer sequences encoding a specific heptapeptide cleavage recognition site (ENLYFQS) for the nuclear-inclusion-a (NIa) proteinase of tobacco etch virus (TEV). The protein coding sequences used were: a Trichoderma harzinum endochitinase, a truncated NIa proteinase of TEV, and a wheat oxalate oxidase. When p35S-CNO construct was tested in Arabidopsis thaliana, the polyprotein was properly cleaved after translation and the products exhibited functional enzymatic activity in vivo.

Arabidopsis↗
PubMed2006

Enzymatic characterization of a trypsin-like serine protease encoded by the genome of cell fusing agent virus.

Cell fusing agent virus (CFAV) is a positive strand RNA insect virus first isolated from a mosquito cell line. Based on viral morphology, phenotypic and phylogenetic studies, CFAV had been tentatively assigned to the genus Flavivirus (family Flaviviridae). The determination of the CFAV polyprotein complete sequence showed a putative serine protease domain analogue to the flaviviral NS2B/NS3 complex. This complex had been extensively studied, because it represented one of the main targets for antiflavivirus therapy development. We report herein the biochemical characterization of CFAV DeltaNS2B-NS3pro protease complex. CFAV polyprotein sequence was computationally analysed to identify the amino-acid regions involved in protease activity. We designed, expressed and purified a catalytically active protease whose enzymatic properties were determined using fluorogenic substrates. Our results showed that, despite the low level of conservation of its amino-acid sequence, CFAV protease exhibited physico-chemical properties of other flaviviruses (high pH value requirement for optimal activity, inhibition by salt and preference for substrates featuring a basic residue at P(1) position).

Amino Acid Sequence↗
PubMed2026

Transcriptome mining and comparative genomics reveal 36 putative novel marafivirus species and conserved evolution of the marafibox regulatory element.

BACKGROUND: Marafiviruses are plant-infecting RNA viruses associated with several economically important crops, but their genomic diversity remains incompletely characterized. OBJECTIVE: This study aimed to identify previously unrecognized marafivirus genomes and investigate their genomic features and evolutionary relationships. METHODS: Publicly available plant transcriptome datasets were systematically mined to detect marafivirus-like sequences. Recovered genomes were analyzed using comparative sequence analysis, phylogenetic reconstruction, and genome organization characterization. RESULTS: A total of 62 marafivirus-like genomes were recovered from 33 independent sources representing diverse plant hosts. Polyprotein-based comparative and phylogenetic analyses grouped these genomes into 36 lineages likely representing novel species. All newly identified viruses clustered within the Marafivirus clade. Genome organization analysis revealed conserved polyprotein architecture and widespread presence of the marafibox promoter element. Conservation of additional open reading frames among closely related isolates aided identification of potentially functional genes. CONCLUSION: These findings substantially expand the known diversity of marafiviruses and demonstrate the effectiveness of transcriptome mining for discovering previously unrecognized plant viruses.

Phylogeny↗