Complete nucleotide sequence of RNA 3 from alfalfa mosaic virus, strain S

We report the sequence of RNA 3 from strain S of Alfalfa mosaic virus (2,055 nucleotides). This RNA codes for a 32.4 kd protein (P3) and for the 24 kd coat protein (P4). The largest part of the sequence was established using RNA sequencing methods. The completion of the sequence in the region coding for P3 was achieved with cloned cDNA synthesized after priming at internal sites of RNA 3. Comparison of the RNA sequences coding P3 and P4 proteins in strain S with those reported in the literature for strain 425 revealed a higher amino acid substitution rate (3%) for P3 than for P4 (congruent to 1%) despite a similar average base substitution of 3-4% in these regions. In P3, two out of nine amino acid changes occur in hydrophilic regions. The amino acid changes in P4 do not modify the local hydrophilicity distribution. The intercistronic region displays a low degree of base substitution (2%) when compared with the untranslated 3'-end region (3.6%) or the 5'-end leader region (8%), the average substitution rate being 3.2%.     

The complete nucleotide sequence of the potexvirus white clover mosaic virus.

The complete nucleotide sequence (5845 nucleotides) of the genomic RNA of the potexvirus white clover mosaic virus (WC1MV) has been determined from a set of overlapping cDNA clones. Forty of the most 5'-terminal nucleotides of WC1MV showed homology to the 5' sequences of other potexviruses. The genome contained five open reading frames which coded for proteins of Mr 147, 417, Mr 26,356, Mr 12,989, Mr 7,219 and Mr 20,684 (the coat protein). The Mr 147,417 protein had domains of amino acid sequence homology with putative polymerases of other RNA viruses. The Mr 26,356 and Mr 12,989 proteins had homology with proteins of the hordeivirus barley stripe mosaic virus RNA beta and the furovirus beet necrotic yellow vein virus (BNYVV) RNA-2. A portion of the Mr 26,356 protein was also conserved in the cylindrical inclusion proteins of two potyviruses. The Mr 7,219 protein had homology with the 25K putative fungal transmission factor of BNYVV RNA-3.     

The nucleotide sequence of cowpea mosaic virus B RNA

The complete sequence of the bottom component RNA (B RNA) of cowpea mosaic virus (CPMV) has been determined. Restriction enzyme fragments of double-stranded cDNA were cloned in M13 and the sequence of the inserts was determined by a combination of enzymatic and chemical sequencing techniques. Additional sequence information was obtained by primed synthesis on first strand cDNA. The complete sequence deduced is 5889 nucleotides long excluding the 3' poly(A), and contains an open reading frame sufficient to code for a polypeptide of mol. wt. 207 760. The coding region is flanked by a 5' leader sequence of 206 nucleotides and a 3' non-coding region of 82 residues which does not contain a polyadenylation signal.     

The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein.

The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus. To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography. The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity. In addition, beta(b) bound RNA without detectable sequence specificity. However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates. Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites. These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 1.

Double-stranded cDNA of alfalfa mosaic virus (AlMV) RNA 1 has been cloned and sequenced. From clones with overlapping inserts, and other sequence data, the complete primary sequence of the 3644 nucleotides of RNA 1 was deduced: a long open reading frame for a protein of Mr 125,685 is flanked by a 5'-terminal sequence of 100 nucleotides and a 3' noncoding region of 163 nucleotides, including the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common. The two UGA-termination codons halfway RNA 1, that were postulated by Van Tol et al. (FEBS Lett. 118, 67-71, 1980) to account for partial translation of RNA 1 in vitro into Mr 58,000 and Mr 62,000 proteins, were not found in the reading frame of the Mr 125,685 protein.     

Complete nucleotide sequence of alfalfa mosaic virus RNA3.

A full-length cDNA clone of alfalfa mosaic virus (AMV) RNA3 was prepared and sequenced. The 2,037 base sequence contains two open reading frames of 903 and 666 nucleotides that code for a 32,400 dalton protein (32.4K protein) and the 24,380 dalton coat protein, respectively. A 5'-noncoding sequence of 240 bases preceeding the 32.4K protein contains homologous regions that may have a function in its translation. The intercistronic junction is 49 bases long, the last 36 bases representing the 5'-end of the subgenomic RNA4. The remaining 179 bases comprise the 3'-terminal noncoding sequence.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 4.

Alfalfa mosaic virus RNA 4, the subgenomic messenger for viral coat protein, was partially digested with RNase T1 or RNase A and the sequence of a number of fragments was deduced by in vitro labeling with polynucleotide kinase and application of RNA sequencing techniques. From overlapping fragments, the complete primary sequence of the 881 nucleotides of RNA 4 was constructed: the coding region of 660 nucleotides (not including the initiation and termination codon) is flanked by a 5' noncoding region of 39 nucleotides and a 3' noncoding region of 182 nucleotides. The RNA sequencing data completely confirm the amino acid sequence of the coat protein as deduced by Van Beynum et al. (Fur.J. Biochem. 72, 63-78, 1977).     

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end. This coding region is flanked by a 5'-terminal sequence of 54 nucleotides and a 3'-noncoding region of 166 nucleotides which includes the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common.     

黄瓜花叶病毒山东株（CMV—SD）RNA2全和cDNA克隆的构建及全序列分析

分别利用５’ＲＡＣＥ和３’ＲＡＣＥ确定了ＣＭＶ－ＳＤＲＮＡ２的５’和３’未端序列,在此基础上,利用ＲＴ－ＰＣＲ得到了ＲＮＡ２的５’端一半的ｃＤＮＡＤＱ ＢＴＧ Ｐｃ２５和３’端一半的ｃＤＮＡ克隆ＰＣ２３,并通过拼接构建了ＲＮＡ２全长ｃＤＮＡ克隆ＰＣ２Ｆ。     

Comparison of barley stripe mosaic virus strains

BSMV (barley stripe mosaic virus) particles were obtained in a pure state from infected host plant tissues of Hordeum vulgare. The three genomic parities (alpha, beta and gamma) were amplified by PCR using specific primers for each particle; each was cloned. Partial sequence of the alpha, beta and gamma segments was determined for the Egyptian isolate of barley stripe mosaic virus (BSMV AE1). Alignment of nucleotide sequences with that of other known strains of the virus, BSMV type strains (CV17, ND18 and China), and the generation of phylogenetic trees was performed. A low level of homology was detected comparing 467 bp of the a and 643 bp of the segments to that of the other strains, and thus BSMV alpha and beta segments were in separate clusters. However, 1154 bp of the gamma segments of BSMV AE1 showed a high level of homology especially to strain BSMV ND18, as they both formed a distinct cluster. Northern blotting of pure BSMV AE1 virus and H. vulgare-infected tissue were compared using an alpha ND18 specific probe. Western blotting using antibodies specific for the coat protein (CP) and the triple gene block 1 (TGB1) protein, which are both encoded by the beta ND18 segment, still indicated a high level of similarity between proteins produced by BSMV ND18 and AE1. We suggest that the BSMV AE1 isolate is a distinct strain of BSMV which reflects the genetic evolutionary divergence among BSMV strains and members of the Hordeivirus group.     

Nucleotide sequence of the 3''-terminal tRNA-like structure in barley stripe mosaic virus genome.

This paper describes the sequence of 257 nucleotides from the 3' end of RNA 2 of barley stripe mosaic virus ( BSMV , strain Argentina Mild) including an internal oligo (A) tract localized at a distance of 236 nucleotides from the 3' end, and the 3' terminal tRNA-like structure accepting tyrosine. This sequence is shown to be the same with RNAs 1,2 and 3 of another BSMV strain, Norwich , for at least the first 106 nucleotides from the 3' end. The 3' extremity of BSMV RNA bears some resemblance to tRNATyr from yeast in its primary structure. The possible secondary structures of the tRNA-like sequence in BSMV genome are discussed.     

The structure of barley stripe mosaic virus double-stranded RNAs

The terminal structures of the double-stranded replicative forms (RFs) of barley stripe mosaic virus (BSMV) RNAs 1–3 have been investigated. All three BSMV RFs have identical right-hand ends but unique left-hand ends. The plus (+) strands of RFs lack the 3′-ultimate A typical for the encapsidated BSMV RNAS. The 3′-termini of the minus (−) strands contain an unpaired G. It was demonstrated that the internal poly(A) tract of BSMV genome has an equivalent poly(U)-counterpart in the RF (−) strands. The possible role of these peculiarities of BSMV RF structure in RNA replication is discussed.     

Primary structure of RNA 3 of barley stripe mosaic virus and its variability

The complete nucleotide sequence was determined for three variants of the third genomic component of BSMV strain Argentina mild. The common variant, RNA 3 (2797 nucleotide), contains two open reading frames (ORFs) coding for two proteins with Mr of 74,229 (putative BSMV RNA polymerase) and Mr of 16,994. The second ORF is expressed from a subgenomic RNA. The extended variant RNA 3 differs from the common one only by the presence of a direct tandem repeat 351-363 nucleotides in length (with some variability) encompassing part of the leader sequence and the beginning of the first ORF. The resulting protein has a Mr of about 86,000. The defective variant, RNA 4, carries a deletion of 185 nucleotides in the 3'-end proximal part of the first ORF, which shortens the product to a Mr of 60,344.     

Molecular basis of plant viral virulence; the complete nucleotide sequence of an attenuated strain of tobacco mosaic virus.

The total genome sequence of L11A, an attenuated strain of tobacco mosaic virus (TMV), has been determined. This strain is able to multiply in tomato plants without inducing any remarkable symptoms, but to protect them from later infection with virulent TMV strains. When compared with the recently published total genome sequence of TMV L (the virulent ancestral strain of L11A) ten base substitutions were found in the L11A genome. Seven of these occurred in the third letters of in-phase codons and did not influence amino acids. Only three, which were in the common reading frame for both the 130K and 180K proteins, resulted in amino acid changes. Together with the result of the partial sequence of RNA of L11, an intermediate strain in sequential isolation from L to L11A, it is observed that one base at the nucleotide position 1117 is changed from L to L11 and two bases at the positions 2349 and 2754 are changed from L11 to L A11.