Investigation of the complexity of barley stripe mosaic virus RNAs with recombinant dna clones

RNA isolated from the Type, ND18, and Norwich strains of barley stripe mosaic virus (BSMV) was electrophoresed in agarose gels, transferred to nitrocellulose, and hybridized with BSMV-specific complementary DNA (cDNA) or recombinant DNA clones derived from ND18 RNA. Genomic RNA components 1 (Mr = 1.43 x 10(6)) and 2 (Mr = 1.24 x 10(6)) were resolved in all three strains, but RNA 3 (Mr = 1.1 x 10(6)) was seen only in the ND18 and Norwich strains. A low-molecular-weight RNA (Mr = 0.27 x 10(6)), thought to be a subgenomic (SG) RNA, was also detected in RNA preparations from all three strains by staining with toluidine blue or ethidium bromide and by hybridizing with cDNA or selected recombinant DNA probes. Three classes of recombinant DNA clones, designated pBSM1, pBSM2, and pBSM3, were identified by hybridization of nick-translated recombinant DNA to electrophoretically separated viral RNAs. Clones in the pBSM1 class hybridized only to RNA 1 of all three strains and class pBSM2 clones hybridized only to RNA 2 of all three strains. Class pBSM3 clones hybridized to RNA 3 of the ND18 and Norwich strains and to RNA 2 of the Type strain, but not to RNA 2 of ND18 or Norwich. Based on the sizes of the BSMV-specified inserts in clones designated pBSM1a, pBSM2a, and pBSM3a, we estimate that a minimum of 44, 63, and 63% of the nucleotide sequences of ND18 and Norwich RNAs 1, 2, and 3, respectively, are unique. Furthermore, because the combined size of the inserts in pBSM2a and pBSM3a is approximately 15% greater than the estimated size of RNA 2, it is probable that the second RNA component of the Type strain actually consists of two RNA species which are similar in size but have different sequences. The SG RNA component is viral specific and contains sequences common to clones derived from RNA 3.     

Comparative analysis of polypeptides synthesized in vivo and in vitro by two strains of barley stripe mosaic virus

Proteins synthesized in a wheat germ system containing RNA from the Type or ND18 strain of barley stripe mosaic virus (BSMV), or polyribosomes from Type or ND18-infected barley, were compared with each other and with polypeptides from Type and ND18-infected plants. Polypeptides with apparent molecular weights (Mr) of 25, 67, and 120 x 10(3) that were induced in infected barley were synthesized in vitro by polyribosomes from infected plants or RNA from purified BSMV. The 25,000 Mr polypeptide was identified as BSMV coat protein by coelectrophoresis with coat protein from purified virions, by immunoprecipitation, and by absence of [35S]methionine incorporation into the protein. Strain-specific differences were observed in two additional translation products synthesized in both wheat germ and reticulocyte lysate systems containing Type of ND18 RNA. RNAs from both strains directed synthesis of 71 and 82 x 10(3) Mr polypeptides. However, ND18 RNA directed synthesis of a larger amount of the 71,000 Mr polypeptide, whereas with Type RNA the 82,000 Mr polypeptide predominated. These two proteins may be processed in vivo because reduced synthesis of both polypeptides in wheat germ extracts containing polyribosomes from infected plants was correlated with increased synthesis of the 67,000 Mr polypeptide. Several additional polypeptides synthesized in the wheat germ system were probably premature termination products because their synthesis in the reticulocyte lysate was greatly reduced.     

A comparative study on the in vitro translation products of individual RNAs from two-, three-, and four-component strains of barley stripe mosaic virus

Passaging through plants of the three-component barley stripe mosaic virus (BSMV) strain Norwich (Norwich III) yielded a two-component isolate (Norwich II). A study was made of the sets of polypeptides translated in a rabbit reticulocyte and wheat embryo cell-free systems from individual RNAs of (1) the natural three-component strain Norwich III, (2) a two-component isolate derived from the former (Norwich II), and (3) an isolate intermediate between the three- and two-component ones, Norwich;. Translation of RNA 1 from all three variants of the Norwich strain in vitro yields a single major product with a molecular weight (Mr) of 120,000 (p120). RNA 2 from Norwich III in vitro produces two polypeptides: the viral coat protein (p23) and, in certain ionic conditions, a polypeptide of 25,000 M(r) (p25). RNA 3 from Norwich III is translated into a protein with Mr of 75,000 (p75). Conversion of Norwich III into Norwich II is accompanied by the changes in the coding specificity of RNA 2; beside the coat protein and p25, a protein of 85,000 M(r) (p85) is formed upon its translation-a feature characteristic of RNA 2 of the naturally occurring bipartite BSMV strains (Russian, type). With the Norwich(i) variety, which is marked by a significantly reduced portion of RNA 3 in the total virion RNA preparation, RNA 2 in addition to the coat protein, p25, and p85 directs the synthesis of another product with M(r) of about 78,000. Thus, in conversion of the three-component BSMV into a two-component one, the loss of RNA 3 is concomitant with the actuation in RNA 2 of a sequence coding for p85. RNAs 1-3 of the quadripartite Argentina Mild (AM) BSMV strain code in vitro for the same polypeptides as RNAs 1-3 of Norwich III. AM RNA 4 is translated as a monocistronic template into a polypeptide with Mr of 55,000 (p55). Amino acid sequences of p85, p75, and p55 are shown to overlap among these polypeptides but not to appreciably overlap with p120 sequences. Data presented here allowed a tentative structure to be proposed for genome of two-, three-, and four-component BSMV strains.     

Nucleotide sequence and genetic organization of barley stripe mosaic virus RNA gamma

The complete nucleotide sequences of RNA gamma from the Type and ND18 strains of barley stripe mosaic virus (BSMV) have been determined. The sequences are 3164 (Type) and 2791 (ND18) nucleotides in length. Both sequences contain a 5'-noncoding region (87 or 88 nucleotides) which is followed by a long open reading frame (ORF1). A 42-nucleotide intercistronic region separates ORF1 from a second, shorter open reading frame (ORF2) located near the 3'-end of the RNA. There is a high degree of homology between the Type and ND18 strains in the nucleotide sequence of ORF1. However, the Type strain contains a 366 nucleotide direct tandem repeat within ORF1 which is absent in the ND18 strain. Consequently, the predicted translation product of Type RNA gamma ORF1 (mol wt 87,312) is significantly larger than that of ND18 RNA gamma ORF1 (mol wt 74,011). The amino acid sequence of the ORF1 polypeptide contains homologies with putative RNA polymerases from other RNA viruses, suggesting that this protein may function in replication of the BSMV genome. The nucleotide sequence of RNA gamma ORF2 is nearly identical in the Type and ND18 strains. ORF2 codes for a polypeptide with a predicted molecular weight of 17,209 (Type) or 17,074 (ND18) which is known to be translated from a subgenomic (sg) RNA. The initiation point of this sgRNA has been mapped to a location 27 nucleotides upstream of the ORF2 initiation codon in the intercistronic region between ORF1 and ORF2. The sgRNA is not coterminal with the 3'-end of the genomic RNA, but instead contains heterogeneous poly(A) termini up to 150 nucleotides long (J. Stanley, R. Hanau, and A. O. Jackson, 1984, Virology 139, 375-383). In the genomic RNA gamma, ORF2 is followed by a short poly(A) tract and a 238-nucleotide tRNA-like structure.     

Sequence comparison of the 3' ends of a subgenomic RNA and the genomic RNAs of barley stripe mosaic virus

All strains of barley stripe mosaic virus examined encapsidate small amounts of an 800-nucleotide (NT) gamma-subgenomic (sg) RNA. This sgRNA has been isolated from genomic (g) RNAs of the Type and North Dakota 18 (ND18) strains and the sequence of these RNAs has been compared near the 3' end. The immediate 3' termini of the gRNAs terminate in the icosomer-GGUCCCCCAAGGGAAGACCAOH-3' and differ from the sgRNAs, which are polyadenylated. The poly(A) tracts of the sgRNAs are heterogeneous with lengths ranging from 10 to greater than 150 NT. Polyacrylamide gel electrophoresis of complementary (c) DNAs transcribed in the presence of dideoxynucleotides reveals that the sgRNAs from Type and ND18 have almost identical sequences for at least 160 NT adjacent to the 5' side of the poly(A) region. This region of the sgRNA from the ND18 strain is nearly identical to a 95-NT sequence adjacent to a poly(A) tract located at the 3' end of a 2050-base pair cDNA cloned from the gamma-genomic RNA of ND18. These results suggest that the sequences encoding the sgRNA are located upstream of an internal poly(A) region situated more than 200 NT from the 3' end of the gamma-genomic RNA.     

Multicomponent properties of barley stripe mosaic virus ribonucleic acid

RNA extracted from purified barley stripe mosaic virus (BSMV) was analyzed by density gradient centrifugation and polyacrylamide gel electrophoresis. All strains tested had a prominent 21.3 S RNA, which was separated into two components by gel electrophoresis. Most preparations of North Dakota 18 and Argentina Mild strains had an additional 19.5 S component, which was resolved into one or two RNA species by gel electrophoresis. Some preparations also contained variable amounts of two or three minor RNA components migrating slowly in gels; these components sedimented at about 30 S in sucrose gradients. The two 19.5 S components were more variable than other components, and occasionally one or both disappeared during virus subculture. Double-stranded RNA isolated from infected plants migrated more slowly in polyacrylamide gels than did single-stranded RNA isolated from purified virions, but both types of RNA from the same strain had the same number of major RNA components. The RNAs of BSMV migrated more slowly in gels than expected from their sedimentation rates if brome mosaic virus RNAs and barley ribosomal RNAs were used as standards, which suggests that BSMV RNAs and the standard RNAs differ in secondary configuration. Mixtures of the separated RNA components were more infectious than single components, which indicates that more than one RNA component was necessary to establish infection.     

Nucleotide sequence of barley stripe mosaic virus RNA alpha: RNA alpha encodes a single polypeptide with homology to corresponding proteins from other viruses

The complete nucleotide sequence of RNA alpha from the Type strain of barley stripe mosaic virus has been determined. The RNA is 3768 nucleotides long and contains a single open reading frame which codes for a polypeptide of 1139 amino acids (mw 129,634). The open reading frame is flanked by a 5'-terminal sequence of 91 nucleotides and a 3'-nontranslated region composed of a short poly(A) tract followed by a 238-nucleotide tRNA-like structure. The amino acid sequence of the polypeptide (alpha a) encoded by the open reading frame has homology with the TMV 126K protein and with related polypeptides from other viruses. The carboxy-terminal portion of the alpha a polypeptide also has limited homology with the 58K (beta b) protein encoded by BSMV RNA beta and includes a consensus sequence found in mononucleotide-binding polypeptides.     

The presence of a cap structure at the 5'-end of barley stripe mosaic virus RNA

About 70% of the molecules in the total RNA preparation isolated from barley stripe mosaic virus (BSMV, Norwich strain) were capped, i.e., contained a 5'-terminal inverted 7-methylguanosine. At least two individual components of the genomic RNA (RNA 1 and RNA 3) were capped. Thus the RNA components of BSMV contain both a 3'-poly(A) sequence (A. A. Agranovsky et al., Virology91, 95-105, 1978) and a 5'-terminal cap.     

Complexity of the Argentina mild strain of barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) is known to have unusual strain-specific variations in the number of high molecular weight RNAs that can be resolved by gel electrophoresis. Analysis with recombinant DNA clones has revealed that all strains contain three distinct hybridizing species of RNA, which we now designate alpha, beta and y. The alpha- and beta-RNAs are about 4200 and 3650 nucleotides (NT), respectively, but the gamma-RNAs vary in size depending on the strain. The gamma-RNA of the Type strain is 3650 NT, and is difficult to resolve from beta-RNA by gel electrophoresis. However, the gamma-RNA of the North Dakota 18 (ND18) strain is 3250 NT and is clearly separated from beta-RNA. The results presented below show that the Argentina mild (AM) strain contains a mixture of gamma-RNAs of 3650, 3250, and 2900 NT. The 3650- and 3250-NT gamma-RNAs appear to be functional because AM subclones containing either 3650- or 3250-NT gamma-RNA can be isolated from plants inoculated with low concentrations of virus. The 2900-NT gamma-RNA is probably not capable of functioning as a genomic RNA because this RNA is not a component of any of the AM subclones.     

Intraviral homology and subgenomic RNAs of pepper ringspot virus

The Tobraviruses constitute a group of rod-shaped, bipartite, plus-stranded RNA viruses. We report on homologies between the two viral genomic RNA molecules of pepper ringspot virus (PRV) and on the subgenomic components generated from them during infection. It has previously been shown that the 3'-terminal 459 nucleotides of PRV RNA-1 and RNA-2 are identical. Here it is shown that there is a second homology between RNA-1 and a region of RNA-2, located at least 240 nucleotides downstream from its 5' terminus. In agreement with strains in another Tobravirus group, we observe three subgenomic components in extracts of plants infected with PRV. Two of these, RNAs-1a (1.6 kb) and -1b (0.8 kb), are derived from RNA-1 (the larger genomic RNA). They are probably mRNAs for 29- and 16-kDa nonstructural virus proteins. The smaller genomic RNA, RNA-2, generates the subgenomic component RNA-2a (1.3-1.45 kb) which is probably an efficient capsid protein mRNA. Previously reported subgenomic components of 2.8 and 1.1 kb are shown to be electrophoretic artifacts. RNA-2a, but not 1a or 1b, is present in virion RNA preparations, indicating that it is the only subgenomic species to be encapsidated.     

Antigenic and molecular analysis of influenza A(H3N2) virus strains isolated in 1985 in open and closed communities of northern Germany.

Antigenic and molecular analyses of influenza A(H3N2) virus strains isolated in 1985 during outbreaks in open and closed communities of North Germany were carried out. The data obtained have shown that 11 strains isolated in a closed orphanage were antigenically similar to each other. The electrophoretic mobilities of either HA, NP, M1 and NS1 polypeptides or of double stranded RNA segments were indistinguishable. Analysis of viruses isolated at the same time from open communities has revealed that they contained at least three groups of strains differing in homology of 3-5 RNA segments. These data support the idea that an outbreak of influenza in a community is caused by single virus strain, from which their slightly different variants of the virus arise during circulation among sensitive persons.     

Nucleotide sequence of barley stripe mosaic virus RNAα: RNAα encodes a single polypeptide with homology to corresponding proteins from other viruses

The complete nucleotide sequence of RNAα from the Type strain of barley stripe mosaic virus has been determined. The RNA is 3768 nucleotides long and contains a single open reading frame which codes for a polypeptide of 1139 amino acids (mw 129,634). The open reading frame is flanked by a 5′-terminal sequence of 91 nucleotides and a 3′-nontranslated region composed of a short poly(A) tract followed by a 238-nucleotide tRNA-like structure. The amino acid sequence of the polypeptide (αa) encoded by the open reading frame has homology with the TMV 126K protein and with related polypeptides from other viruses. The carboxy-terminal portion of the as polypeptide also has limited homology with the 58K (βb) protein encoded by BSMV RNAβ and includes a consensus sequence found in mononucleotide-binding polypeptides.     

Stability of expression of Neisseria gonorrhoeae lipooligosaccharides.

We compared multiple lipooligosaccharide (LOS) extracts from individual strains of Neisseria gonorrhoeae. Each of the extracts was prepared from single mass cultures grown on solid media under similar conditions but separated by time. We found only subtle variations in the number, electrophoretic mobility, and concentration of components of the LOSs from individual strains. We found no variation in the expression of a 3.6-kilodalton LOS component that carries the L8 LOS epitope. A significant variation in the 3-deoxy-D-manno-octulosonic acid content was found among different extracts from the same strain, but this variation appeared to be unrelated to the other LOS characteristics studied.     

Highly purified cucumber mosaic virus-induced RNA-dependent RNA polymerase does not contain any of the full length translation products of the genomic RNAs

The extensively purified RNA-dependent RNA polymerase (RNA replicase) induced by infection of cucumber seedlings with cucumber mosaic virus (CMV) was investigated to determine whether the enzyme contains full-length translation products of the CMV genomal RNAs. Two experimental approaches were used. (1) RNA replicase preparations from plants infected with three strains of CMV (Q, P, and T) all had almost identical polypeptide compositions, which included a major polypeptide of relative mobility on sodium dodecyl sulfate-polyacrylamide gels of Mr, 100,000, and two other proteins, Mr 110,000 and Mr, 35,000, present in smaller, varying amounts. These polypeptides were unique to enzyme preparations from CMV-infected plants and had similar electrophoretic mobilities to the translation products of Q-CMV RNAs 1, 2, and 3, respectively. Analysis of the in vitro translation products of the corresponding RNAs of the other two strains of CMV showed, however, that those of P-CMV RNA 2 and T-CMV RNAs 1 and 3 varied significantly in size from the translation products of the corresponding Q-CMV RNAs. (2) Peptide mapping studies, using digestion with the V8 protease from Staphylococcus aureus or cleavage with CNBr, confirmed that none of these three components of the highly purified RNA replicase was a translation product of the CMV RNAs. The work reported in this paper, therefore, showed that the full-length translation products of the genomal RNAs of CMV were absent from the RNA replicase induced by this virus after solubilization and extensive purification.     

In vitro translation of cucumoviral satellites. I. Purification and nucleotide sequence of cucumber mosaic virus-associated RNA 5 from cucumber mosaic virus strain S

The satellite cucumber mosaic virus (CMV)-associated RNA 5 (CARNA 5) of CMV strain S (CMV-S) which previously had been assigned the capability both to direct the synthesis of two small proteins in vitro (R. A. Owens and J. M. Kaper, 1977, Virology, 80, 196-203) and to induce the tomato necrosis disease in the presence of its helper virus (J. M. Kaper and H. E. Waterworth, 1977, Science, 196, 429-431), has been reinvestigated. Polyacrylamide gel electrophoretic analyses under partially denaturing conditions of CARNA 5 preparations from CMV-S grown in tobacco reveal a mixture of three distinct RNA species which have been isolated and partially characterized. In order of decreasing electrophoretic mobility they have been designated RNA 5, (n)CARNA 5, and (S)CARNA 5, respectively. RNA 5 has been partially sequenced and shown to represent 3'-terminal fragments of the CMV genomic RNAs. (n)CARNA 5 is responsible for the tomato necrosis-inducing properties of the mixture and coelectrophoreses with tomato necrosis-inducing CARNA 5 from CMV strain D. (S)CARNA 5 does not cause tomato necrosis; its complete nucleotide sequence was determined and is compared here to the published sequences of the CARNA 5s of four other CMV strains. A companion paper (M. J. Avila-Rincon et al., 1986, Virology, 152, 455-458) provides unequivocal evidence that the in vitro translation of nonnecrotic (S)CARNA 5 produces two small polypeptides resembling those described earlier.     

Partial sequence comparison of eight new Chinese strains of hepatitis E virus suggests the genome sequence is relatively stable

Partial genomic sequences representing 420 nucleotides of a nonstructional region, 480 nucleotides of the putative RNA polymerase region, and 540 nucleotides of the structural region of epidemic-associated Chinese strains of hepatitis E virus (HEV) were obtained by direct sequencing of PCR-amplified DNA. Comparison with previously published HEV sequences showed a clear relatedness of all Chinese strains to each other and to a Pakistani strain (Sar-55). All eight Chinese strains examined had very similar sequences (98.5-99.8% homology) in the regions examined and were much closer to the Pakistani strain (Sar-55) (97.9-98.4% homology) than to the Burmese strain (92.5-93.3% homology). Sequence comparisons of the three genomic regions in the Chinese strains indicated that the RNA polymerase region was much more conserved than the other nonstructural region or the structural region. HEV isolates from three remote geographic regions of China had sequences closely related to each other.     

The nature of the polypeptide encoded by each of the 10 double-stranded RNA segments of reovirus type 3

Under suitable conditions of denaturation, the double-stranded (ds) RNA segments of reovirus can be translated in cell-free protein synthesizing systems. Since all 10 segments of reovirus ds RNA can be isolated in virtually pure form, this provides a means for determining the nature of the polypeptide encoded by each individual segment. The complete coding assignment set was determined for the Dearing strain of reovirus serotype 3. Polypeptide identification was made not only on the basis of electrophoretic migration rates in both the phosphate- and Tri-glycine (Laemmli)-based polyacrylamide gel systems, but also on the basis of comparing peptide profiles of in vitro translation products and authentic reovirus polypeptides after digestion with staphylococcal V8 protease. The latter method provides absolute identification. The assignment set is (using the commonly accepted designation for the ds RNA segments, but a newly proposed nomenclature for the polypeptides): Segment L1 codes for the minor virion component λ3, and segments L2 and L3 code for the two major virion core components λ2 and λ1, respectively; segment M1 codes for a minor virion component μ2, segment M2 codes for the polypeptide that is present in virions both in the form of the minor component μ1 and as the major component μ1C which is derived from it by cleavage, and segment M3 codes for the nonstructural polypeptide μNS; and segment S1 codes for the minor outer capsid shell component σ1, segment S2 codes for the core component σ2, segment S3 codes for the nonstructural polypeptide σNS, and segment S4 codes for the major outer capsid shell component σ3.     

Structural and biological characteristics of reoviruses isolated from Muscovy ducks (Cairina moschata)

Two Muscovy duck reoviruses, strains y1/79 and 1625/87, were investigated with regard to their genome organization, polypeptide pattern, serotype specificity, and pathogenicity. Electrophoretic analysis of the genome revealed the migration pattern of avian reoviruses. In spite of general conformity, great polymorphism was detected in the electrophoretic mobility of individual genome segments of the two strains sharing only three segments of identical size (L1, M2, M3). Only one segment (M3) migrated into the same position as the corresponding segment of prototype chicken reovirus S1133. The basic electrophoretic mobility pattern of the immunopre‐cipitated polypeptides, eight structural and two non‐structural, closely resembled that of the chicken reovirus. However, considerable strain‐specific variation was also seen at the protein level, with the σ_c polypeptides exhibiting the most obvious migration differences. Based on the results of cross‐neutralization assays the two Muscovy duck reovirus strains were grouped into one serotype, with no cross‐reactivity to the chicken serotype S1133. In experimental infections, despite virus replication proved by faecal virus excretion and antibody response, only strain y1/79 was pathogenic for 16‐day‐old Muscovy ducklings, thus making strain 1625/87 a possible candidate as vaccine strain.     

Structure of the 3' extremity of barley stripe mosaic virus RNA: evidence for internal poly(A) and a 3'-terminal tRNA-like structure

The results of a previous study suggested that the poly(A) sequence in barley stripe mosaic virus (BSMV) RNA is intercalated between a 3'-terminal tyrosine-accepting structure and the 5'-terminal coding part of the BSMV genome. Here we show that poly(A)+ and poly(A)- fractions of BSMV RNA can be cleaved into two fragments specifically at the position of poly(A) or oligo(A) sequence with RNase H from Escherichia coli in the presence of oligo(dT)10. The shorter fragment (Sh) retains the ability of intact viral RNA to be aminoacylated, i.e., it represents the 3'-terminal part of BSMV RNA. Electrophoretic analysis of Sh-RNA reveals three closely positioned subspecies with an average length of about 210 nucleotides. The long 5'-terminal RNA fragment (L) produced by RNase H treatment has electrophoretic mobility similar to that of intact BSMV RNA, but displays neither amino acid-accepting ability nor infectivity. Nevertheless, L-RNA possesses the same messenger activity as the intact viral RNA and codes for the same pattern of polypeptides in rabbit reticulocyte lysate in vitro translation assays.