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 of the cowpea and bean strains of southern bean mosaic virus

Features of the primary structure and translation of the genomic RNAs of the cowpea and bean strains of southern bean mosaic virus have been investigated in order to assess the similarity of the two viruses. The sequence of 400 bases at their 3' termini have been determined. These include the 3' noncoding regions and extend well into the coat protein cistrons. The noncoding regions (136 bases for the cowpea strain RNA and 129 bases for the bean strain RNA) show no obvious sequence homology. However, extensive base as well as amino acid sequence homology exists in the coding region. RNAs from both strains have a small protein attached to their 5' terminus-the protein in the cowpea strain being the smaller of the two. In vitro studies show that there are similarities in the overall mode of translation of the genomes of the two viruses. Although corresponding proteins are synthesized they differ in size.     

A comparative study of the alkaline disassembly of two strains of tobacco mosaic virus

Exposure of tobacco (Tb) and tomato (Tm) isolates of tobacco mosaic virus (TMV) to dilute alkaline solutions (pH > 8.0) at 0 degrees results in disassembly of the virus particles. Over the range of pH and NaCl concentration studied, Tm-TMV was more sensitive to alkaline conditions than was Tb-TMV. Kinetic analysis demonstrates that both isolates disassemble in a stepwise manner and that each produces six major intermediate-size particles.     

Sequence relations and coding properties of a subgenomic RNA isolated from barley stripe mosaic virus

A subgenomic (SG) RNA ( approximately 800 nucleotides) is a minor component of barley stripe mosaic virus RNAs. The SG-RNAs isolated from the Type and North Dakota 18 (ND18) strains of BSMV have sequence homology with RNA 2 of the ("pseudo-two component") Type strain, which has two electrophoretic components, but only limited homology is evident with RNA 2 of the ND18 and Norwich strains, which have three electrophoretic components ("three component" strain). Instead, eDNAs from SG-RNA hybridize most efficiently with RNA 3 of the ND18 and Norwich strains. In wheat germ extracts the SG-RNAs direct the synthesis of two polypeptides with apparent molecular weights of 20 to 21 x 10(3). However, these two polypeptides were difficult to detect by polyacrylamide gel electrophoresis of extracts from Type- or ND18-infected barley and so appear not to accumulate during infection.     

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.     

Aminoacylation of barley stripe mosaic virus RNA: polyadenylate-containing RNA has a 3'-terminal tyrosine-accepting structure

Barley stripe mosaic virus (BSMV) RNA which was previously reported to contain poly(A) sequences (Agranovsky et al., 1978) can be specifically esterified with tyrosine in vitro in the presence of an aminoacyl-tRNA synthetase fraction from wheat embryos. All the three RNA components of the BSMV strain with a three-component genome (Norwich) and both RNA components of a two-component strain (Russian) can be tyrosylated. The poly(A)-containing (bound to oligo(dT)-cellulose) and poly(A)-deficient (not bound to oligo(dT)-cellulose) fractions of BSMV RNA display a similar amino acid-accepting ability. The nucleotide sequence which accepts tyrosine is coupled with the intact genomic polyadenylated BSMV RNA. The viral RNA isolated after sucrose density gradient centrifugation under drastic denaturing conditions retains its aminoacylating activity, which suggests that this activity is not due to the presence in a BSMV RNA preparation of a tyrosine tRNA associated with BSMV RNA. Inhibition of aminoacylation of the 3'-oxidized (treated with sodium metaperiodate) BSMV RNA suggests that the tyrosine-accepting structure is localized at the 3' terminus of BSMV RNA molecules. It is shown that segments of different lengths obtained upon random fragmentation can be tyrosylated. The 3'-terminal (tyrosine-accepting) poly(A)+ segments can be isolated. The shortest segments of viral RNA capable of being aminoacylated [i.e., containing both tRNA-like structure and poly(A)] consists of approximately 150-200 nucleotides. The analysis of the oligonucleotides derived from individual BSMV RNA components labeled with (32)P at the 3' end revealed two types of 3'-terminal sequences different from poly(A). It is suggested that a poly(A) sequence is intercalated between a 3'-terminal tyrosine-accepting structure and the 5'-terminal portion of poly(A)+ BSMV RNA.     

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.     

In vitro translation products of turnip crinkle virus RNA

Turnip leaves infected with turnip crinkle virus accumulate a 35-kd polypeptide which comigrates with the major protein from isolated virions. RNA from TCV virions directs the synthesis of a number of polypeptides in vitro including a 35-kd protein which is immunoprecipitable with antiserum to virus particles. Translation of viral RNA size-fractionated on sucrose gradients or methyl mercurichydroxide-containing gels shows that the TCV coat protein is synthesized primarily from RNA fragments which are smaller than the genomic RNA. A satellite RNA species found in virions does not direct the synthesis in vitro of any identifiable protein.     

Amino acid sequence in the proteolytic glycopeptide of Barley stripe mosaic virus

Gel filtration of a Pronase digest of purified BSMV capsid protein yielded a fraction containing demonstrable carbohydrate in association with a tripeptide. The amino acid sequence of this tripeptide was determined to be Gly-Asp-Ala. The carbohydrate was associated with aspartic acid. Amide nitrogen determination of the glycopeptide showed one residue of ammonia liberated per residue of aspartic acid, suggesting the carbohydrate moiety is attached through the amide bond to asparagine.     

Differences in polyadenylate length between individual barley stripe mosaic virus RNA species

Barley stripe mosaic virus (BSMV) RNA which has been shown to contain an internal polyadenylate sequence (Agranovsky, Dolja, Gorbulev, Kozlov, and Atabekov, "Virology", 113, 114-187, 1981; Agranovsky, Dolja, and Atabekov, "Virology," 119, 51-58, 1982) was resolved into oligo(dT)-cellulose-bound [poly(A)+] and unbound [poly(A)-] fractions. Individual RNAs of all strains tested (i.e., those with two-, three-, or four-components) differentially responded to affinity chromatography. Thus, in all cases the poly(A)+ fraction was dramatically enriched in RNA 1 whereas RNA 2 was found mostly in the poly(A)- fraction. RNA 3 was distributed approximately equally between poly(A)+ and poly(A)- fractions. The bulk of RNA 4 was in the poly(A)- fraction. The poly(A) length spectra in total, poly(A)+, and poly(A)- BSMV RNA fractions were compared. The length of poly(A) in total BSMV RNA was 8 to 40 residues. Fragments of 19 to 40 residues and probably longer were predominant in the poly(A)+ RNA fraction. The poly(A)- fraction contained tracts having not more than approximately 25 adenylate residues, with the bulk of the tracts having 12 or fewer residues.     

Translational studies with turnip yellow mosaic virus RNAs isolated from major and minor virus particles

The major and minor nucleoprotein components in turnip yellow mosaic virus (TYMV) preparations have been fractionated by four cycles of CsCl gradient equilibrium centrifugations. RNAs isolated from the various fractions were translated in a rabbit reticulocyte cell-free system. Three classes of RNAs were studied: genomic RNA with an Mr of 2.0 x 10(6), the subgenomic coat protein messenger with an (r) of 2.4 x 10(5) (H. Guilley and J. P. Briand, 1978, Cell15, 113-122) and RNA molecules of intermediate sizes. Genomic RNA is found in the major TYMV components. These particles do not contain the coat protein messenger. The latter messenger has mainly been detected in two types of minor components, those which have an RNA content exceeding that of the major TYMV virions and particles which contain the coat protein messenger as their sole RNA constituent. RNAs of intermediate sizes are found in minor components less dense than the major particles; the full-length translational products of these RNAs and of genomic RNA overlap with one another and share a common NH2 terminus. It is concluded that a number of intermediate-sized RNAs and the genomic RNA have a common site for initiation of translation located close to their 5'-termini.     

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.     

Sequential encapsidation of heterologous RNAs with papaya mosaic virus protein.

Papaya mosaic virus protein encapsidates heterologous RNAs under conditions normally restricted to the encapsidation of homologous RNA if the heterologous RNAs are first initiated with protein under conditions of nonspecificity. This means that initiation, but not maturation, controls the specificity of encapsidation.     

Altered balance of the synthesis of plus- and minus-strand RNAs induced by RNAs 1 and 2 of alfalfa mosaic virus in the absence of RNA 3

The synthesis of viral plus-strand and minus-strand RNAs in cowpea protoplasts inoculated with mixtures of alfalfa mosaic virus nucleoproteins (B, M, Tb, and Ta) was analyzed by the Northern blotting technique. A mixture of B, M, and Tb induced the synthesis of plus-strand RNAs 1, 2, 3, and 4 and three minus-strand RNAs corresponding to RNAs 1, 2, and 3, respectively. Compared to this complete infection, a mixture of B and M induced the synthesis of a reduced amount of plus-strand RNAs 1 and 2 and a greatly enhanced amount of minus-strand RNAs 1 and 2. No detectable viral RNA synthesis was induced by mixtures of B and Tb or M and Tb. It is concluded that expression of genomic RNAs 1 and 2 results in the formation of a replicase activity that produces roughly equal amounts of viral plus- and minus-strand RNAs and that an RNA 3-encoded product, possibly the coat protein, is responsible for a switch to an asymmetric production of viral plus-strand RNA. The observation that no minus-strand corresponding to the subgenomic RNA 4 is produced suggests that recognition of the genome segments by the viral replicase involves sequences outside the 3'-terminal regions that are homologous to RNA 4.     

Cross-protection and interference between electrophoretically distinct strains of cucumber mosaic virus in tomato

Cucumber mosaic virus (CMV) was partially purified by polyethylene glycol precipitation from extracts of tomato leaflets. The presence and quantity of two strains with different electrophoretic mobilities was analyzed by polyacrylamide gel (2.5%) electrophoresis of preparations from as little as 50 mg of tissue. This method was used to analyze interference and cross-protection between the strains. Coinoculation resulted in local and systemic mixed infections and reductions in the synthesis of both strains. Systemic symptoms and accumulation of the strain with the more severe symptoms were not detected in up to 13 newly formed leaves in five of six plants preinfected with the mild strain and challenged with the severe strain. Systemic accumulation of the mild strain was not detected in each of six plants preinfected with the severe strain and challenged with the mild strain. Both strains could accumulate in challenge-inoculated leaves. The level or absence of interference in these leaves did not affect systemic cross-protection. Cross-protection between these strains of CMV involves considerable inhibition of virus accumulation in addition to absence of symptoms.     

Okra mosaic virus empty protein shells in nuclei

Okra mosaic virus produces large quantities (approximately 10 mg/g of tissue) of empty viral protein shells in inoculated cucumber cotyledons. Virus particles could not be detected in isolated nuclei, but empty protein shells accumulated there. At one day after inoculation, about half the total viral protein shells were found in the nucleus. This accumulation occurred in the presence of virus particles in the cytoplasm. The most likely explanation for this active and preferential accumulation is that viral coat protein enters the nucleus in the form of monomers or pentamers and hexamers, and is assembled into shells once inside.     

Viral protein synthesis in barley protoplasts inoculated with native and fractionated brome mosaic virus RNA

When barley protoplasts were inoculated with brome mosaic virus (BMV) RNAs 1 and 2, there was a pronounced synthesis of the 110,000- and 100,000-dalton virally coded proteins. In contrast, there was no detectable synthesis of any viral proteins following inoculation with RNA 3 alone or RNA 4 alone. When RNAs 1 and 2 were recombined with RNA 3 in the inoculum, the profile of proteins synthesized was identical to that following inoculation with similar quantities of unfractionated BMV RNA; i.e., the 35,000-dalton virally coded protein and coat protein were synthesized in addition to the two high-molecular-weight viral polypeptides. RNAs 1 and 2 were shown not to be selectively bound (in preference to RNAs 3 or 4); hence, these data reveal that one or both of these RNAs encode proteins involved in early events of infection, perhaps replication.     

Replication of tobacco mosaic virus. VII. Further characterization of single- and double-stranded virus-related RNAs from TMV-infected plants

The single-stranded (ss) and double-stranded (ds) RNAs produced in tobacco tissue as a result of infection by tobacco mosaic virus (TMV) have been reinvestigated. 32P-labeled probes consisting of either cDNA or viral RNA, complementary to specific regions of either the viral RNA or its negative strand, respectively, were used in "Northern" hybridization experiments. Of the 10 ssRNA bands observed, all but four appeared to be artifacts of electrophoresis. These four RNAs were found on polyribosomes and are presumed to be true mRNAs; three were identified as the well-known genomic RNA, the I2-mRNA and the coat protein mRNA, or LMC. The fourth RNA species of MW approximately 1.2 x 10(6) had not previously been specifically identified as a subgenomic RNA of TMV. The viral RNA which gave rise to the six artifactual ssRNA bands was heterogeneous in size and was shown to be encapsidated in vivo. Upon electrophoresis, these heterogeneous RNA fragments comigrated approximately with plant rRNAs also present in the extracts, generating the observed artifactual bands. Four dsRNAs were also identified. From molecular weight and hybridization analyses, they appeared to be double-stranded forms of the above four polyribosome-associated ssRNAs. Attempts to translate proteins from the denatured dsRNAs in vitro were unsuccessful. A population of low-molecular-weight, TMV-specific ssRNAs, (+) and (-) in sequence, was generated during infection; however these RNAs were believed to be breakdown products.