Tobacco mosaic virus protein synthesis is correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis

The synthesis rates of three proteins of tobacco mosaic virus (TMV), 160, 110, and 17.5 kDa, were monitored at intervals after interruption of synthesis of TMV RNA. Following inhibition of synthesis of both single-stranded and double-stranded RNAS by shifting wild type TMV to 40° or is mutant III₂-35 to 35°, the synthesis rates of viral proteins declined sequentially, with that of the larger proteins declining faster. When viral RNA synthesis was prevented with cordycepin, synthesis rates of the 110 and 160-kDa proteins declined rapidly, while the 17.5-kDa protein decreased more slowly. These data imply that the functional mRNA is transitory, probably nascent RNA, and that each protein is produced independently. The process of translation of viral mRNA was not temperature sensitive and occurred normally for brief periods after shift to restrictive temperatures. When single-stranded RNA synthesis was inhibited differentially from double-stranded RNA synthesis, protein synthesis was correlated with double-stranded RNA synthesis and not single-stranded RNA synthesis. Following a shift of is mutant IV-35 to 35°, a shift that immediately stopped single-stranded RNA synthesis without inhibiting double-stranded RNA synthesis, all three viral proteins continued to be produced normally. Also, after return of wild type TMV to 25° after a 1-hr incubation at 40°, viral protein and double-stranded RNA synthesis recovered in parallel to the normal rate after 8 hr whereas single-stranded RNA synthesis, which had been reduced more drastically, recovered more slowly after 16 hr.     

Genetically engineered Tobacco mosaic virus as nanoparticle vaccines

Tobacco mosaic virus (TMV) is an RNA virus that typically infects plants but has recently been adapted for vaccine development, owing to the suitability of the virions for modifications as nanoparticles. TMV also has a simple functional structure of a 6.4 Kb (+)-strand RNA encapsidated by a single coat protein, which permits facile genetic manipulation. In this review, we describe recent advances in the manipulation of TMV for the development of several different types of vaccines, including ones that induce antibody and T-cell responses that are protective in pathogen and tumor challenge animal models. Lastly, we describe how TMV self-assembly properties are being used to make a new mammalian RNA pseudovirus, that has unique characteristics for RNA and protein antigen delivery to antigen-presenting cells.     

In vivo interaction between Tobacco mosaic virus RNA-dependent RNA polymerase and host translation elongation factor 1A

Several host translation elongation factors have been suggested to play essential roles in the replication and translation of viral RNAs in plants, animals and bacteria. Here, we show the interaction between eukaryotic translation elongation factor 1A (eEF1A) and Tobacco mosaic virus (TMV) RNA-dependent RNA polymerase (RdRp) in vivo by immunoprecipitation. The tobacco eEF1A interacted not only with 3'-untranslated region (3'-UTR) of TMV RNA but also directly with RdRp without mediation by the 3'-UTR. The methyltransferase domain of TMV RdRp was indicated to be responsible for the interaction with eEF1A in vitro and in yeast. These results suggest that eEF1A is a component of the virus replication complex of TMV.     

Molecular variability of Tobacco vein banding mosaic virus populations

The incidence of Tobacco vein banding mosaic virus (TVBMV) on tobacco increases dramatically in China recently and it has caused great economic losses. To gain insights into the evolutionary mechanisms of TVBMV, a total of 40 TVBMV isolates were collected from different tobacco production regions in China and their genomic regions encoding helper component-proteinase (HC-Pro), the third protein (P3), the first 6K protein (6K1) and coat protein (CP) were sequenced. Phylogenetic analyses revealed that TVBMV isolates can be divided into two evolutionary divergent groups based on P3, the frame-shifting pipo and 6K1 genes, and three groups on HC-Pro and CP genes. The populations from most parts of mainland China (MC) showed frequent gene flow; those from Yunnan province in south western China always formed a separate group (YN) and also had frequent within-group gene flow. However, the gene flow between groups MC and YN was uncommon. Our results revealed that all the tested TVBMV genes were under negative selection and the HC-Pro gene was under the strongest constraints. Recombination events were identified in 13 of the 42 analyzed isolates. This study suggested that negative selection, gene flow and recombination were important evolutionary factors driving the genetic diversification of TVBMV.     

Effects of replacing the movement protein gene of Tobacco mosaic virus by that of Tomato mosaic virus

The broad bean strain of Tobacco mosaic virus (TMV-B) infects Nicotiana tabacum White Burley systemically whereas the tomato strain of T. mosaic virus (ToMV-S1) induces necrotic local lesions and is restricted to inoculated leaves. To examine the possible role of the viral movement protein (MP) in these symptom differences, a chimaeric virus (T/OMP) was produced in which the TMV-B MP gene was replaced by the ToMV-S1 MP gene. T/OMP induced the same symptoms as TMV-B in N. tabacum White Burley. However, in N. tabacum Samsun NN and other plants containing the N resistance gene, T/OMP caused necrotic lesions that were smaller than those produced by TMV-B but similar in size to those of ToMV-S1. We conclude that ToMV MP gene can substitute functionally for the TMV-B MP gene, and that the MP gene influences the size of necrotic local lesions on N-containing hosts.     

Aminoacylation and messenger functions of eggplant mosaic virus RNA

The predominant RNA of eggplant mosaic virus (EMV) was found to have a molecular weight of 1.9 x 10(6) by formamide gel electrophoresis. Electrophoretic analysis of virion protein under dissociating conditions revealed two polypeptides, a major component of 21,000 daltons, and a minor component of 22,000 daltons. Both peptides were present in translation products coded by RNA isolated from virions, but the proportion of the 22,000-dalton peptide was higher in products synthesized using RNA isolated from EMV-infected Datura leaves as messenger. Since the viral RNA showed a marked tendency to aggregate, it is possible that these polypeptides were translated from trace amounts of small EMV RNAs present as contaminants of the 1.9 x 10(5)-dalton genomic RNA. Although the addition of tRNA from infected or healthy Datura leaves, or from wheat germ, stimulated amino acid incorporation, no changes were discerned in the profile of cell-free translation products after electrophoretic separation. Nonaminoacylated and valylated EMV RNA stimulated similar levels of amino acid incorporation, and the translation products appeared identical. Valine bound to genomic EMV RNA was not donated during protein synthesis.     

Infectivity of partially encapsidated tobacco mosaic virus RNA

Tobacco mosaic virus was reconstituted in vitro, and the infectivity of intermediate products at various stages of reconstitution was investigated in relation to their rod length. The infectivity of intermediates was exponentially related to their rod length throughout the reconstitution process, indicating that no particular region of TMV-RNA is preferentially susceptible to inactivation or more important for infection.     

Tobacco mosaic virus particles contain ubiquitinated coat protein subunits

Virions of tobacco mosaic virus (TMV) are composed of a single strand of RNA, encapsidated in about 2130 copies of a coat protein of MW 17,500. Asselin and Zaitlin [Virology 91, 173-181 (1978)] demonstrated that virion preparations also contained small amounts of a second protein of MW 26,500, which they termed "H protein." H protein, detectable to an average frequency of one per virion, was thought to be a protein of host origin. Subsequent studies [Collmer, Vogt, and Zaitlin, Virology 126, 429-448 (1983)] showed the H protein was comprised of a backbone of TMV coat protein, linked by a postulated isopeptide bond to a small protein that probably was of host origin. The host-derived moiety of H protein is shown here to be ubiquitin, most probably coupled to the coat protein at lysine 53. This finding is based on microsequencing of the H protein, and is substantiated by immunoblotting analysis with antibodies to human ubiquitin. Conjugated ubiquitin was detected in virions of all five strains of the virus tested. To our knowledge, this is the first report of a ubiquitinated viral structural protein.     

Purification and characterization of brome mosaic virus RNA-dependent RNA polymerase

RNA-dependent RNA polymerase (RdRp) was solubilized from cellular membranes of brome mosaic virus (BMV)-infected barley. The solubilized enzyme was subsequently purified by glycerol gradient centrifugation and DEAE ion-exchange chromatography. The purified enzyme proved to be highly stable and both dependent on and specific for BMV RNAs. The enzyme is inhibited by high template RNA concentrations. This inhibition indicates feedback regulation of minus-strand synthesis. The nonstructural viral protein P1 was found to be a component of the RdRp complex (R. Quadt, H.J.M. Verbeek, and E.M.J. Jaspars, 1988, Virology 165, 256-261). Using antibodies directed against a C-terminal peptide of P1 a complex of seven 125I-labeled proteins was precipitated. This indicates that the P1 protein is associated with at least six proteins in the infected cell.     

Nucleotide sequence and translation of satellite tobacco mosaic virus RNA

Satellite tobacco mosaic virus (STMV) is a plant virus with a 17-nm icosahedral particle encapsidating a 0.3 X 10(6) Mr ssRNA genome that depends on tobamoviruses for its replication. The complete nucleotide sequence of STMV RNA deduced in the experiments described here was 1059 nucleotides in length. The efficiency of labeling viral RNA with [gamma-32P]ATP using T4 polynucleotide kinase was not affected by treatment with tobacco acid pyrophosphatase and/or bacterial alkaline phosphatase, indicating that the majority of the 5' termini of encapsidated STMV RNAs were not phosphorylated. The 240 3'-terminal nucleotides of STMV RNA and either tobacco mosaic virus (TMV) U1 RNA or TMV U2/U5 RNA had greater than 65% overall sequence similarity, with two nearly identical regions of 40 and 50 bases, respectively. There were no other regions of sequence relatedness to TMV RNA. The 19 5'-terminal nucleotides of STMV RNA had greater than 65% sequence similarity with the 16 5'-terminal nucleotides of brome mosaic virus (RNA 3 and 50% sequence similarity with the 12 5'-terminal nucleotides of the Q strain of cucumber mosaic virus RNA 3. The first open reading frame (ORF) beginning at base 53 encoded a 6800 Mr protein that corresponded in size to a major in vitro translation product directed by STMV RNA. A second ORF, beginning at nucleotide 163, had the capacity to code for a protein that corresponded in size (17,500 Mr) to the other major in vitro translation product. The first 12 codons of this ORF corresponded to the sequence of the N-terminal amino acids of the capsid protein. Western-blot analysis of the in vitro translation products revealed that the 17,500 Mr protein had the same electrophoretic mobility as the authentic capsid protein; it was also antigenically related to the capsid protein, but the 6800 Mr protein was not. Time course analysis of in vitro translation demonstrated that the 6800 Mr protein was synthesized at the same time as the capsid protein and did not arise by the proteolytic cleavage of a larger precursor polypeptide. These results suggest that the genome of STMV functioned as a polycistronic messenger RNA. It has not been determined if the 6800 Mr protein is synthesized in vivo. STMV RNA had untranslated regions of 52 and 418 nucleotides at its 5' and 3' termini, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Does tobacco mosaic virus RNA contain cytokinins?

Tobacco mosaic virus was previously reported to contain between 5 and 17 modified nucleosides with cytokinin activity per RNA molecule. Using a new and highly sensitive method for gas chromatography of permethylated cytokinins, we were unable to detect cytokinins in enzymic or alkaline digests of TMV RNA. The method was sensitive enough to assay 1 cytokinin nucleoside per 10 TMV RNA molecules. Soybean callus bioassay also failed to detect activity in TMV RNA hydrolysates which corresponded to any of the cytokinin nucleosides known to occur in RNA. We therefore suggest that TMV RNA does not contain cytokinin nucleosides.     

Cloning and sequencing of peach rosette mosaic virus RNA1

The complete nucleotide sequence of peach rosette mosaic nepovirus (PRMV) RNA1 has been determined. A grapevine isolate of PRMV from Michigan was propagated and purified and cDNA clones representing 99. 5% of the RNA1 were constructed. The cDNA and direct RNA sequence analysis revealed a RNA species of 8004 nucleotides, excluding a 3' polyadenylated tail. The 5'- and 3'-untranslated regions were 52 and 1474 nucleotides, respectively. Computer analysis of the PRMV RNA1 nucleotide sequence unveiled a single long open reading frame of 6477 nucleotides, which is capable of encoding a 240 kDa polyprotein. Analysis of the predicted amino acid sequence of RNA1 revealed amino acid motifs characteristic of a replicase, proteinase, NTP-binding protein and a proteinase cofactor. The order and identity of these putative proteins are consistent with other nepoviruses. Analysis of PRMV RNA1 further distinguishes the taxonomic subdivisions within the nepovirus group, confirms the subgroup three status of PRMV and lays the groundwork for a replicase-mediated resistance strategy.     

Sequence and organization of southern bean mosaic virus genomic RNA

The genomic RNA sequence of the cowpea strain of southern bean mosaic virus (SBMV-C) has been determined. The genome is 4194 nucleotides in length and has four open reading frames. A 5' proximal open frame, from base 49 to base 603, corresponds to the length of the P4 proteins translated in cell-free extracts from full-length and smaller virion RNA. The largest open frame extends from base 570 to base 3437 and encodes the two largest proteins translated in cell-free extracts from full-length virion RNA. Segments of this open reading frame's predicted amino acid sequence resemble those of known viral RNA polymerases, ATP-binding proteins, and viral genome-linked proteins. A third open frame extends from base 1895 to base 2380 and has not been correlated with an in vitro translation product. The fourth open reading frame is located in the 3' terminal region of the genome extending from base 3217 to base 4053. This frame encodes the SBMV capsid protein which is translated from subgenomic, virion RNA.     

Molecular epidemiology of Cucumber mosaic virus and its satellite RNA

Molecular analysis of viral isolates can yield information that facilitates an understanding of virus epidemiology and has been termed molecular epidemiology. This approach has only recently been applied to plant viruses. Results on the molecular epidemiology of Cucumber mosaic virus (CMV) and its satellite RNA (satRNA) in Spain, where CMV is endemic in vegetable crops are presented here. To characterise the genetic structure of CMV populations, c. 300 isolates, representing 17 outbreaks (i.e. sub-populations) in different crops, regions and years, were compared. Genetic analyses of CMV isolates were done by ribonuclease protection assay of cRNA probes representing RNA1, RNA2 and the two open reading frames in RNA3. All isolates belonged to one of three genetic types: Sub-group II and two types of Sub-group I. The genetic structure of the 17 sub-populations varied randomly, without correlation with location, year, or host plant species. Thus, CMV in Spain shows a metapopulation structure with local extinction and random recolonisation from local or distant virus reservoirs. The frequency of mixed infections and of new genetic types generated by reassortment of genomic segments or by recombination was also estimated. Results indicate that heterologous genetic combinations are not favoured. About 30% of CMV isolates were supporting a satRNA. The frequency of CMV isolates with a satRNA differed for each sub-population, being c. 1 in eastern Spain in 1990 and decreasing to c. 0 in distant regions and in subsequent years. Molecular analyses of CMV-satRNA isolates show high genetic diversity, due both to the accumulation of point mutations and to recombination. The CMV-satRNA population is a single, unstructured one. Thus, the CMV-satRNA population has a genetic structure and dynamics different from those of its helper virus. This indicates that CMV-satRNA has spread epidemically on the extant virus population from an original reservoir in eastern Spain. The relevance of these results for the control of CMV infections is discussed.