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.     

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.     

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.     

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.     

Translation of tick-borne encephalitis virus (flavivirus) genome in vitro: Synthesis of two structural polypeptides

RNA isolated from tick-borne encephalitis virus (TBEV) was translated in an mRNA-dependent cell-free system derived from Krebs-2 cells, producing a set of polypeptides with M_r ranging from ca. 13,000 to 160,000 and higher. Two of these polypeptides with M_r of 13,000 (p13) and 53,000 (p53) were identified as TBEV core polypeptide V2 and the polypeptide moiety of envelope glycoprotein, V3, respectively. The amino acid sequences of p13 and p53 were also shown to be contained in a high-molecular-weight polypeptide (M_r ～ 118,000). The label from the initiator tRNA species, f-[³⁵S]Met-tRNA_f^Met, could be incorporated into p13 but not into p53, suggesting that p13 is encoded in a region of the viral genome immediately adjacent to the site at which the translation in vitro is initiated; on the other hand, the p53 coding segment appears to be located further from the initiation point. The two structural polypeptides of TBEV were accumulated in vitro with dissimilar kinetics and both differed from the majority of other translation products in that their synthesis was relatively resistant to an increase in KCl concentration in the incubation mixture. Possible modes of synthesis of TBEV structural proteins and post-translational processing of their precursor are discussed.     

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.     

Adenovirus-associated virus polypeptides synthesized in cells coinfected with either adenovirus or herpesvirus.

One major and two minor structural polypeptides of adenovirus-associated virus (AAV) were synthesized in AAV-infected cells coinfected with either adenovirus or herpesvirus as helper. The molar proportions of these polypeptides appeared to be the same as those found in the virion. Transport of these polypeptides from the cytoplasm to the nucleus occurred rapidly. No evidence was found for the synthesis of a large precursor protein in either system. Since complete AAV virions are not found in AAV-infected cells coinfected with herpesvirus, these findings indicate a failure in virus assembly in this system. This failure may be expressed at the level of DNA strand segregation or encapsidation of DNA, or in faulty capsid assembly.     

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.     

Comparative cytology of nine isolates of cauliflower mosaic virus

The cytology of nine isolates of cauliflower mosaic virus was compared by light and electron microscopy. Differences noted among the isolates were: the size of cytoplasmic inclusion bodies, frequency of virions occurring free in the cytoplasm as opposed to those in inclusions, the ratio of virions to the amount of matrix protein in inclusions, and intraplastidial inclusions induced by one of the isolates. It was concluded that these four cytological features are expressions of the viral genome rather than the host. One of the isolates induced different sized inclusions in two different hosts.     

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.     

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.     

Immunoprecipitable polypeptides specified by varicella-zoster virus

Polypeptides encoded by varicella-zoster virus (VZV) in infected cell cultures have been identified by radioimmune precipitation techniques. Detergent-solubilized extracts of VZV-infected cells were reacted with highly specific VZV antisera raised in strain-2 guinea pigs immunized with sonicates of syngeneic virus-infected cells. Fractionation of the immunoprecipitates in acrylamide slab gels demonstrated an average of 16 polypeptides, which ranged in molecular weight from 32,000 to å200,000. These included the three major immunogenic glycoproteins (gp 62, gp 98, and gp 118) and a prominent higher molecular weight nonglycosylated polypeptide at 155,000. One of the [³⁵S]methionine-labeled polypeptides comigrated with purified actin. Not all polypeptides were visible in any one particular fluorogram, but comparative analysis of polypeptide profiles derived from electrophoreses performed with different gel concentrations and different crosslinkers (methylene-bisacrylamide and N,N-diallyltartardiamide) clearly established a consistent and reproducible pattern of radioactive bands. A low background of radio-activity was nonspecifically precipitated by the antigen-antibody-protein A complexes; however, with the exception of a common band comigrating with actin, the electrophoretic profiles representing virus-specific and nonspecific immunoprecipitates were easily distinguished.     

Influence of the host cell on proteins synthesized by different strains of influenza virus.

The synthesis of influenza virus proteins has been studied quantitatively in nine different virus-cell systems, using three strains and three cell lines. The systems were remarkable for their variation. The only common properties to emerge were that NS₁ and NP were always synthesized earlier than M and HA₀ and secondly, with the exception of the P proteins, proteins were synthesized in amounts proportional to their time of appearance, i.e., the first protein to appear was present in the greatest amount. Otherwise variations were found in the order of appearance of newly synthesized proteins, in the relative amounts synthesized, and in the total synthesized. These variations were unrelated to virus strain or cell type, to the yield of infectious virus, or to its infectivity: HA ratio, and to the shut-off of host protein synthesis. We conclude that the pattern of protein synthesis in a particular virus-cell system is unique.     

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.     

Intermediates of the in vitro assembly and disassembly of southern bean mosaic virus

Southern bean mosaic virus (SBMV) was swollen by treatment with EDTA at pH 7.5 and dissociated into RNA and protein in 1 M NaCl. Aliquots of this preparation were diluted with appropriate buffers to obtain samples in varying concentrations of NaCl, and components of these samples were sedimented through sucrose solutions and dissolved in 0.01 M Tris-HCI buffer, pH 7.5. The protein content and sedimentation properties of components in these preparations were determined. When the NaCl molarity in the treatment exceeded 0.6 M the preparations contained RNA with approximately six protein subunits per SBMV RNA molecule. The protein content of the preparations increased from 30 protein subunits per RNA molecule to 145 protein subunits per RNA molecule as the NaCl molarity used in the treatment was decreased from 0.5 to 0.1 M. The positions of sedimentation of components in these preparations in density gradient centrifugation were intermediate between those of RNA and EDTA-swollen virus. The sedimentation rate of these assembled components increased as the NaCl molarity used in the treatment was decreased. Similar components were assembled when preparations of RNA and protein dissociated from SBMV by dialysis in neutral buffers containing EDTA and 1 M NaCl were diluted to lower NaCl molarities. When SBMV was swollen by treatment with EDTA and dissociated in various concentrations of NaCl, the components formed were similar to those obtained by assembly in the same NaCl molarities. Preparations in the pH 7.5 buffer contained single components which sedimented at 56 S, 55 S, 54 S, 51 S, 46 S, 38 S, 33 S, and 24 S. With the exception of the 24 S component, components formed by disassembly in the same NaCl molarities and dissolved in pH 5.0 buffer sedimented faster.     

Comparative examination of the polypeptides of herpes simplex virus: types 1 and 2

Polypeptides synthesized during productive infection of HSV-1 and HSV-2 were found to possess distinct characteristics in regard to localization within the cell, DNA-binding properties, and phosphorylation after synthesis. Continuous labeling for 14 hr or pulse-labeling at successive periods during the replicative cycle with radioactive precursors revealed two types of polypeptide localization: (a) selective accumulation or enhancement within the cytoplasm or nucleus with barely detectable concentrations elsewhere and (b) accumulation in significant concentrations within both cytoplasm and nucleus showing little selective enhancement. Of the polypeptides made during HSV-1 infection 22 were phosphorylated as compared with 16 phosphoproteins specified by HSV-2. Phosphorylation was also implicated in the generation of the four molecular forms comprising the ICP 5–8 complex. Twenty-three polypeptides with affinity for DNA were detected after either type of infection. Sufficient comparisons were made to provide a basis for the tentative listing of 20 polypeptides of HSV-1 with corresponding polypeptides of HSV-2.     

Ultrastructure of mixed plant virus infection: bean yellow mosaic virus with cowpea severe mosaic virus or cowpea mosaic virus in bean

Ultrastructural responses of bean leaf cells simultaneously infected with two morphologically distinct RNA viruses, cowpea mosaic virus (CPMV) and bean yellow mosaic virus (BYMV), or cowpea severe mosaic virus (CSMV) and BYMV, were studied in situ. The major effects on cells infected with two viruses included: (1) association of virus group-specific cytoplasmic inclusions characteristic of each virus; (2) close association of virions into specifically arranged aggregates in which CPMV or CSMV icosahedra were aligned along the long axes of the BYMV rods; and (3) the induced formation of intranuclear inclusions, spheres (22-26 nm in diameter) and filaments (10-14 nm wide and of variable length) in mixed infections of CSMV and BYMV. Intracellular serological testing using ferritin conjugated with CSMV antibodies revealed no relationship between the spherical intranuclear inclusions and CSMV capsids. We conclude that the ultrastructure of mixed infections could be used as another tool for identifying related plant viruses.     

Influenza virus RNA segment 7 has the coding capacity for two polypeptides

The genes of a recombinant human influenza virus A/Japan/305/57:A/Bel/42:A/PR/8/34 have been cloned in bacterial plasmids, and the nucleotide sequence of RNA segment 7 has been determined. The DNA sequence predict two open reading frames, which overlap by 68 bases and have a coding capacity for polypeptides of 27,000 and 11,000 daltons. Segment 7 has been previously assigned as the gene coding for the viral matrix protein of apparent molecular weight 25,000. Therefore gene 7 has the potential to code for a previously unsuspected protein of approximately 11,000 daltons in addition to matrix.     

Ultraviolet inactivation of influenza virus RNA in vitro and vivo.

The uv inactivation of influenza virus RNA within the infected cell indicated that each segment was being transcribed individually. That is, each segment carried its own promoter region. Similar results were obtained when RNA synthesis by uv-irradiated virus was tested in vitro.     

Detection of a wide spectrum of tobacco mosaic virus strains by indirect enzyme-linked immunosorbent assays (ELISA)

The suitability of two ELISA procedures for detecting different strains of tobacco mosaic virus (TMV) by means of a single labeled antibody preparation has been determined. The specificity of the double-antibody sandwich method of ELISA was so great that enzyme conjugates prepared with antibodies to one strain did not react with closely related strains. The indirect method of ELISA which uses an antiglobulin enzyme conjugate was capable of detecting a wide range of TMV strains with antiserum to one strain only. The indirect procedure is thus better suited for diagnostic work. Both methods of ELISA were able to detect relationships between the coat proteins of different TMV strains. These results confirm that the dissociated subunits of different strains are more closely related than the corresponding intact virions.