Nucleotide sequence of pea seed-borne mosaic potyvirus coat protein gene

cDNA of pea seed-borne mosaic potyvirus (PSbMV) RNA was synthesized and cloned in E. coli. Four overlapping clones that cover the complete PSbMV genome, except the extreme 5 terminus, were identified by restriction enzyme mapping, hybridization analysis, and partial sequencing. Overlapping cDNA clones covering 1386 nucleotides of the 3 terminus were sequenced. The nucleotide sequence contains one open reading frame (ORF), followed by an untranslated region of 163 nucleotides and a poly(A)-tract. The deduced amino acid sequence was found to include the C-terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative 49-kD proteinase cleavage site at the N-terminus of the coat protein.     

Nucleotide sequence of the coat protein gene of a necrotic strain of potato virus Y from New Zealand

Summary The sequence of the 3-terminal 1,134 nucleotides of the genome of a New Zealand isolate of a necrotic strain of potato virus Y (PVY^N) has been determined. This sequence contains one large open reading frame of 796 nucleotides, the start of which was not identified, which is capable of encoding a protein of 264 amino acid residues with a molecular weight of 29,631. Comparison of the amino acid sequence with a published coat protein sequence of another strain, PVY-D, and with the amino acid sequence deduced from PeMV cDNA sequence data, confirms that the 3 cistron encodes the viral coat protein in PVY^N. Adjacent to the 3 end of the coding region there is an untranslatable sequence of 326 nucleotides terminating in a polyadenylate tract. An alignment of the PVY^N amino acid sequence with the coat protein sequences of six other potyviruses revealed significant sequence similarities in the internal and carboxy terminal regions. Much amino acid sequence similarity was found between PVY^N, PVY-D, and PeMV (91–93%), suggesting that PeMV should be regarded as a PVY strain. An analysis of the 3-untranslated region of the six potyviruses revealed PVY^N and PeMV as the only viruses displaying sequence similarity in this region. The 3-untranslated sequences of PVY^N and PeMV were further examined for secondary structure.     

Nucleotide sequence of the coat protein gene of pelargonium leaf curl virus and comparison of the deduced coat protein amino acid sequence with those of other tombusviruses

Summary The sequence of 1,787 nucleotides (nts) in the genomic RNA of pelargonium leaf curl virus (PLCV) was determined. It included the entire coat protein (cp) gene (nts 585 to 1,754), 558 nts of the 3 end of the putative RNA polymerase gene, 26 nts of an intercistronic region between the two genes and 33 nts downstream of the stop codon of the cp gene. The cp gene was cloned into the expression vector pET 8c and expressed inE. coli. The deduced cp amino acid sequence of PLCV was compared with those of five other tombusviruses. The closer the degree of serological relatedness between two viruses, the more similarity was found in their cp amino acid sequences not only in the protruding domains, but also in their random and shell domains and in the arm regions. Nucleic acid hybridization tests, cp amino acid comparisons and serological tests all suggest the same order of sequence for the relationships in the tombusvirus group.     

Nucleotide sequence of beet mild yellowing virus RNA

Summary The complete nucleotide sequence of the genomic RNA of beet mild yellowing virus, isolate 2ITB, is reported. The RNA consists of 5722 nucleotides and contains six long open reading frames which conform to the arrangement characteristic of Subgroup 2 luteoviruses. The three 3-proximal open reading frames, which encode the viral coat protein, a putative movement protein and the Readthrough Domain, are highly homologous to the corresponding genes of beet western yellows luteovirus while the three 5-proximal open reading frames are more closely related to the corresponding genes of cucurbit aphid borne yellows luteovirus. The sequence data thus indicate that beet mild yellowing virus should be considered a distinct virus rather than a strain of beet western yellows virus.     

Purification and coat protein gene sequence of a Montana RMV-like isolate of barley yellow dwarf virus

Summary A Montana barley yellow dwarf virus (BYDV) isolate, BYDV-RMV-MT, is serologically identical to the New York RMV type isolate (RMV-NY) but differs in aphid transmission phenotype. A purification procedure for BYDV-RMV-MT was developed and cDNAs encompassing the entire coat protein gene and a portion of the putative polymerase gene of both RMV-MT and RMV-NY were cloned and sequenced. Diameters of RMV-MT virions averaged 24.7 nm. Average virus yield was 4.2 mg/kg plant tissue. There was 81% sequence identity between the clones of MT and NY RMV isolates at the nucleotide level. At the amino acid level the polymerase genes were 91% identical to each other and 74% homologous with that of beet western yellow virus. The coat protein amino acid sequences of the two RMV isolates were only 81% identical and, compared to other sequenced luteoviruses, both were most similar to cucurbit aphid-borne yellows virus.     

Nucleotide sequence and expression in Escherichia coli of the gene encoding the nonstructural protein NCVP2 of bovine rotavirus

Cloned DNA copy of rotavirus genome segment 5 from bovine rotavirus RF strain has been used to determine the nucleotide sequence of the gene that encodes for the nonstructural viral protein NCVP2. The sequence data indicated that segment 5 consists of 1581 base pairs and is A + T rich (66%). The positive strand of segment 5 contains a single open reading frame that extends 491 codons and possesses 5'- and 3'-terminal untranslated regions of 32 and 73 base pairs, respectively. The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 58,654, slightly higher than the apparent molecular weight of NCVP2 (MW 54,000). Although it is not evident whether the gene product is glycosylated, four potential glycosylation sites were found at positions 50, 168, 403, and 438. NCVP2 has been expressed in Escherichia coli using the inducible expression vector pKK233-2. Following IPTG induction high levels of full-length nonfused proteins were synthesized and accumulated in induced cells.     

Nucleotide sequence of cDNA encoding the coat protein of beet yellows virus

A cDNA clone of beet yellows viral RNA expressed the viral coat protein gene inE. coli. The sequence of the 2724 nucleotide insert revealed three open reading frames, the 3 of which was shown to be the coat protein cistron. This cistron is expressed inE. coli, in spite of there being no obvious ribosome binding site upstream.     

Nucleotide sequence of the coat protein coding region of tulip breaking virus

cDNA of tulip breaking virus-tulip (TBV-tulip) RNA was synthesized and cloned inE. coli. One clone that contains a 4.5 kb insert was identified by restriction enzyme analysis, dot immunobinding assay (DIBA), and partial sequencing. Then 1479 nucleotides of the 3-terminus of the clone were sequenced and revealed that the sequence contains one open reading frame (ORF), followed by an untranslated region of 255 nucleotides and a poly(A) tract. The deduced amino acid sequence was found to include the C terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative NIa protease cleavage site at the N terminus of the coat protein.The nucleotide sequence data reported in this paper have been submitted to GenBank nucleotide sequence database and have been assigned the accession number X63630.     

Sequence and identification of the barley yellow dwarf virus coat protein gene

The nucleotide sequence of the coat protein gene of barley yellow dwarf virus (BYDV, PAV serotype) was determined, and the amino acid sequence was deduced. The open reading frame, encoding a protein of relative molecular mass (Mr) 22,047, was confirmed as the coat protein gene by comparison with amino acid sequences of tryptic peptides derived from dissociated virions. In addition, a fragment of this gene expressed in Escherichia coli produced a product which was recognized by antibodies prepared against purified BYDV virions. An overlapping reading frame encoding an Mr 17,147 protein is contained completely within the coat protein gene.     

Nucleotide sequence of the gene encoding the matrix protein of mumps virus (Miyahara strain)

The nucleotide sequence of the gene encoding the matrix (M) protein of mumps virus (MuV), Miyahara strain, has been determined from several overlapping cDNA clones. The M protein mRNA is 1248 nucleotides in length, exclusive of the poly(A) tail, and codes for a protein of 375 amino acids (Mr41,556). Comparison of the deduced amino acid sequence of the M protein of the Miyahara strain with that of the SBL-1 strain revealed that the M proteins of both strains are highly conserved. A significantly lower rate of nucleotide differences conducive to amino acid differences in the M gene compared with other genes appeared to indicate the importance of the conserved primary structure of the M protein for its function.Requests for reprints should be addressed to Kiyoshi Tanabayashi, Department of Measles Virus, National Institute of Health, 4-7-1 Gakuen, Musashimurayama, Tokyo 190-12, Japan.     

Nucleotide sequence of the gene for heat-stable enterotoxin II of Escherichia coli.

Previously, the gene for heat-stable enterotoxin II (STII) has been mapped by transposon Tn5 insertion mutagenesis in the chimeric R-Ent plasmid pCG86 (Mazaitis, A. J., R. Maas, and W. K. Maas, J. Bacteriol. 145:97-105, 1981). DNA segments containing this gene were cloned from the wild-type and STII-insertion-mutant plasmid. The position of the Tn5 insertion was determined, and a 530-base-pair-long segment of the wild-type plasmid corresponding to the Tn5 insertion site was sequenced. An open reading frame for the STII gene was identified and is characterized by typical promoter and ribosome binding site sequences. The deduced STII structural gene codes for a protein 71 amino acids long, including a typical signal peptide of 23 amino acids and a mature protein of 48 amino acids. The size and overall structure of STII are similar to those of STI, but the amino acid compositions of the two heat-stable enterotoxins are completely different.     

Nucleotide sequence of coat protein gene of yam mild mosaic virus, isolated in Papua New Guinea

Summary.  We determined the 3′-termimus 1353 nucleotides (nts) in length excluding the poly (A) tail of yam mild mosaic potyvirus (YMMV) RNA. The sequence starts within a long open reading frame (ORF) 1209 nts and is followed by untranslated region (3′-UTR) of 144 nts. The coat protein (CP) contains 266 amino acids (aa) with molecular ratio (Mr) of approximately 30 kDa. The CP of YMMV differs substantially from yam mosaic virus (YMV), Japanese yam mosaic virus (JYMV) (57 and 61% of amino acid sequence identity) and other potyvirus species. This result suggests that YMMV should be classified as a new yam potyvirus. Received January 29, 1999 Accepted February 26, 1999     

Nucleotide sequences of the coat protein and readthrough protein genes of the Chinese GAV isolate of barley yellow dwarf virus

The nucleotide sequences of the coat protein (CP) and readthrough protein (RTP) genes of the Chinese GAV isolate of Barley yellow dwarf virus (BYDV) were determined. The CP and RTP genes of GAV isolate comprised 600 and 1374 nucleotides, respectively. When the CP and RTP gene sequences of GAV isolate were compared with those of BYDV isolates MAV-PS1, P-PAV, NY-SGV and Cereal yellow dwarf virus RPV (CYDV-RPV), the highest similarity (97.2%) between the CP genes of GAV and MAV-PS1 isolates was observed, while the RTP genes of these two isolates shared a lower similarity (87.8%). The results of the alignment of the deduced amino acid sequences of RTP showed that the sequence diversity observed was located at the C terminus.     

Nucleotide sequence of the coat protein coding region of the potyvirus tobacco vein-banding mosaic virus

Summary The sequence of the 3 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M_r of 30 210. The 3 non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3 non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.     

Nucleotide sequence and in vitro expression of the capsid protein gene of tobacco ringspot virus

The nucleotide sequence of the 3' terminal 2022 nucleotides (nt) of tobacco ringspot virus (TobRV) RNA 2 has been determined. Protein microsequence analysis of the amino-terminal residues of purified capsid protein localized the capsid protein gene between nt 2014 and 583 (from the 3' terminus) of this sequence. The proteolytic cleavage site that is processed to liberate the capsid protein from the RNA 2-encoded polyprotein was identified as Cys-Ala. The predicted translation product from the gene is a 477 amino acid long polypeptide with a calculated MW of 53 kDa. The gene was modified at the 5' end to facilitate sub-cloning, and to provide it with a methionine initiation codon. The modified gene was sub-cloned, transcribed in vitro and expressed in a rabbit reticulocyte lysate translation system, where it directed the synthesis of a 53 kDa polypeptide. Garnier-Osguthorpe-Robson analyses of the secondary structure of the capsid protein predicted the presence of three beta sheet domains, which suggests that this nepovirus capsid may be structurally analogous to those of the como- and picornaviruses. These and other results from computer analyses of the nucleic acid and amino acid sequences, and comparisons with the capsid proteins of nepoviruses and other related viruses are discussed.     

Nucleotide sequence of the coat protein gene of citrus tristeza virus: Comparison of biologically diverse isolates collected in Israel

The sequences of the coat protein genes of four seedling yellows (SY) and four non-SY (NSY) of citrus tristeza virus (CTV) isolates, which were collected in Israel over a period of 30 years, were analyzed. Pairwise comparisons showed extensive similarities in the nucleotide and amino acid sequences of six isolates designated the VT group. This group consists of three NSY isolates that cause a very mild CTV reaction on the sensitive combination of sweet orange (SwO) grafted on sour orange (SO), and three SY isolates that cause severe SY and SwO/SO reactions. MT, a CTV isolate that is consistently nontransmitted byAphis gossypii, was found to be different in two amino acids (Val 103 and Glu 113) from each of theA. gossypii transmissible CTV isolates. Sequencing of the cDNA clones obtained from ST, a variably transmitted CTV isolate, showed extensive sequence variation among the tested clones. The sequence information indicates that the current CTV epidemics in Israel are caused by at least two CTV subspecies (VT and HT) displaying extensive differences in their coat protein genes.     

Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression inEscherichia coli

Total RNA from infectedPhysalis floridana was isolated to generate complementary DNA corresponding to the coat protein (CP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1–3) for fusion protein expression inE. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97–99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRV_cub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.