Translation of tobacco rattle virus RNA in vitro using wheat germ extracts.

When added to extracts from commercial wheat germ, tobacco rattle virus (TRV) RNA stimulated incorporation of radioactive amino acids into protein with an efficiency approaching that of tobacco mosaic virus (TMV) RNA. RNA from the smaller particle (RNA-2) of the CAM strain of TRV was translated largely into a single polypeptide which coelectrophoresed with coat protein and aggregated specifically with unlabeled protein. Coelectrophoresis with coat protein in 3% acrylamide gels indicated a C-terminal sequence in the radioactive product similar to that in coat protein. Attempts to change the pattern of translation products by heating RNA, adding coat protein to incubations, or changing RNA concentration were unsuccessful. RNA from the larger particle (RNA-1) of strain CAM (Campinas) was translated into a variety of products with a maximum molecular weight of 100,000. When mixtures of RNA-1 and RNA-2 were used, RNA-2 was translated preferentially.     

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20–25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3′ terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.     

Genome structure of tobacco rattle virus strain PLB: further evidence on the occurrence of RNA recombination among tobraviruses

RNA-2 (2196 nucleotides) of tobacco rattle virus (TRV) strain PLB was found to consist of a 5'-terminal sequence of 1376 nucleotides identical to the 5'-sequence of RNA-2 of TRV strain PSG and a 3'-terminal sequence of 820 nucleotides that is identical to the 3'-sequence of RNA-1 of strain PLB. Thus, in strains PLB and PSG the same coat protein gene is fused to different RNA-1 derived 3'-termini. By combining RNA-1 of TRV strain TCM with RNA-2 of strain PLB, a viable pseudo-recombinant was formed with genome segments that have nonidentical 3'-sequences. After 25 passages in tobacco each RNA retained its strain-specific 3'-sequences. This indicates that the perfect 3'-homology that occurs between the two genome segments of all natural TRV isolates analyzed so far is not a prerequisite for a stable genotype.     

Expression of the 16K cistron of tobacco rattle virus in protoplasts

An antiserum was raised against a synthetic peptide corresponding to the 18 C-terminal amino acids of a putative 16K protein encoded by the 3'-terminal open reading frame of tobacco rattle virus (TRV) RNA-1. This antiserum was used to demonstrate expression of the 16K cistron in vivo. TRV-infected tobacco protoplasts accumulated similar amounts of 16K protein and viral coat protein but in tobacco plants only the coat protein was detectable. Time course experiments revealed that in protoplasts the accumulation of 16K protein lagged somewhat behind that of coat protein. The 16K protein was incorporated in a high-molecular-weight cellular component that was resistant to treatment with nonionic detergents.     

Expression of the large 5'-proximal cistron of tobacco mosaic virus by 70 S ribosomes during cotranslational disassembly in a prokaryotic cell-free system

In vitro translation of pH 8-washed particles of tobacco mosaic virus (TMV; vulgare, common, or U1 strain) in a cell-free system derived from Escherichia coli (MRE 600) results in a broad size range of TMV RNA-encoded products, including significant amounts of the distinctive 126-kDa polypeptide which is encoded by the first, 5'-proximal open reading frame in TMV RNA and is conventionally expressed by 80 S eukaryotic ribosomes. In an identical prokaryotic translation system, unencapsidated TMV RNA encodes a series of polypeptides of 50 kDa or less. The predominant product of 16-17 kDa frequently appears to comigrate with TMV coat protein. The significance of the former result for mRNA-ribosome interactions and for possible virus-uncoating/early gene expression events within the chloroplasts of infected cells is discussed.     

A tobacco mosaic virus-hybrid expresses and loses an added gene

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3' region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.     

Translation of tobacco rattle virus RNAs in vitro: four proteins from three RNAs

RNAs from the Lisse and PRN strains of tobacco rattle virus were translated in nuclease-treated reticulocyte lysates. With both strains, RNA-1 coded for two proteins of MW 170,000 and 120,000. Addition of 1-2 mM Mg2+ increased the yield of the larger product, apparently by facilitating readthrough of a leaky termination codon at the end of the region coding for the smaller protein. Experiments with yeast suppressor tRNA indicate that this is not a UAG or UAA codon. RNA-2 coded for a protein of the same size as coat protein. A product of MW 30,000 was coded by a third RNA (RNA-3) which in the case of PRN TRV had a molecular weight of 550,000 and could be separated from RNA-2 by gel electrophoresis. Translation of all three RNAs was inhibited by m7GTP. However, RNA-1 also directed a low level of m7GTP-resistant synthesis of a 30,000-dalton protein which appeared to be identical to the RNA-3 product, and it is suggested that RNA-3 corresponds to the 3' end of RNA-1. Thus the strategies of synthesis of the non-coat proteins of tobacco rattle and tobacco mosaic viruses appear to be similar.     

Limitations to tobacco mosaic virus infection of turnip

Summary.  Turnip vein-clearing virus (TVCV) and tobacco mosaic virus (TMV) represent subgroups of tobamoviruses infecting cruciferous and solanaceous plants, respectively. To identify adaptations that may have been necessary in the evolution of the TVCV subgroup from a TMV-like ancestor, the infection of turnip plants by TMV and by chimeras between TMV and TVCV was explored. TMV accumulated at spatially limited sites on inoculated turnip leaves as determined by leaf skeleton hybridization. A plasmid DNA containing a complete TVCV cDNA, when transcribed in vitro, produced RNA that was infectious to tobacco and turnip plants. TVCV-TMV chimeric genomes with junctions within coding regions were not infectious to tobacco, though the movement protein (MP) chimera was infectious to tobacco with a TMV MP transgene. Reciprocal chimeras with junctions between genes were infectious to tobacco. TVCV with a TMV MP gene infected turnips. The other tested chimeras were not detected in non-inoculated leaves, but were found in the inoculated leaves. Thus, the TMV MP is not responsible for the limitation of TMV spread in turnips. Received June 19, 1998 Accepted December 4, 1998     

Enhanced resistance and neutralization of defense responses by suppressors of RNA silencing

The effects of transgenic expression of the potato virus Y (PVY) HCPro silencing suppressor in tobacco were examined on infection by several viruses. Infection by tobacco mosaic virus (TMV) was reduced at 25 °C, but not at 33 °C. By contrast, systemic infection at 33 °C by the TMV expressing green fluorescent protein was promoted by the HCPro. Infection by tobacco rattle virus (TRV) was restricted to local necrotic lesions by the PVY HCPro. However, this resistance was neutralized by expression of the cucumber mosaic virus (CMV) 2b protein from TRV. By contrast, infection by either wild-type CMV or CMV with a deletion of the 2b gene was not affected. Similarly, infection by cauliflower mosaic virus, red clover necrotic mosaic virus (both limited to infection of the inoculated leaves of tobacco) or tomato bushy stunt virus (systemically infecting tobacco) was not altered by the expression of PVY HCPro. Therefore, it appeared that the PVY HCPro was able to induce defense responses at 25 °C, but not at 33 °C, where it actually neutralized a pre-existing defense response. Moreover, the CMV 2b protein was able to neutralize a defense response activated by HCPro in combination with TRV.     

Trans complementation of virus-encoded replicase components of tobacco mosaic virus

We examined whether the 130K and 180K proteins of tobacco mosaic virus (TMV), the putative virus-encoded replicase components, produced by a replication-competent TMV mutant could complement a replication-defective mutant in a single cell. The replication-competent mutant (LDCS29) had a deletion in the coat protein gene and the replication-defective mutant (LDR28) had a large deletion in the gene encoding the 130K and 180K proteins. Neither the replication of LDR28 nor the production of the coat protein from LDR28 or LDCS29 was detected when the mutants were inoculated separately into tobacco protoplasts. However, when the two mutants were co-inoculated, the production of the LDR28 genomic RNA and the subgenomic RNA for the coat protein and accumulation of the coat protein were observed. These results show that the virus-encoded replicase components of TMV complemented the replication-defective mutant in trans.     

Subgenomic components of tobacco rattle virus in infected tissue

Cells infected with the CAM strain of tobacco rattle virus (TRV) contain at least five species of virus-specific RNA in addition to the two genomic RNAs. These species were detected by hybridizing [(32)P]cDNA prepared to TRV RNAs-1 and -2 with electrophoretically separated infected cell RNA species immobilized on diazotized paper. The new viral RNA species have the following apparent molecular weights: RNA-3, 0.5 x 10(6); RNA-4, 0.22 x 10(6); RNA-5, 1.1 X 10(6); RNA-6,0.36 x 10(6); RNA-7, 0.16 x 10(6). RNA-5 is generated from RNA-1 since it hybridizes with RNA-1 cDNA but not with cDNA prepared to RNA-2. The rest hybridize with RNA-2 cDNA and thus are generated from the smaller genomic species.     

Plant virus uncoating as a result of virus-cell wall interactions

The fate of tobacco rattle virus (TRV) particles was studied after leaf panel infiltration. It was found that end-on virus attachment to cell walls of hosts (Nicotiana tobacum L. var. Xanthi-nc) and nonhosts (Zea mays L.) occurred, and that a virus degradative phase commenced immediately after attachment. Length of TRV particles changed drastically following infiltration. The normal length of the particles became smaller than that of the particle carrying the coat protein gene (108 nm). Five days after infiltration, no particles could be detected on the walls of cells bordering intercellular spaces. Virus attachment and degradation was thus shown to be nonspecific. Autoradiographic studies showed that iodinated viral coat protein or a breakdown product of this protein ((125I-label in coat protein of complete virus) and not virus particles are transported to the vascular bundle after infiltration. Tobacco mosaic virus (TMV) and TRV were infiltrated into their respective local lesion hosts. There was an initial time interval early after the infiltration during which wounding (pin pricking) the infiltrated leaf panels resulted in local lesion formation. The results are discussed with regard to the phenomenon of viral genome release in the plant virus infection process.     

Cotranslational disassembly of a cowpea strain (Cc) of TMV: evidence that viral RNA-protein interactions at the assembly origin block ribosome translocation in vitro

Cotranslational disassembly of mixed rod-length populations of tobacco mosaic virus (TMV; vulgare, common or, U1 strain), reveals a reproducible, significant reduction in the level of expression of the intermediate-sized, subgenomic 12-RNA when compared with conventional total viral RNA preparations. I2-RNA encodes a 30-kDa protein, and recent evidence suggests that I2-RNA is unusual in that it lacks a 5'-cap structure. In TMV vulgare, the assembly origin is located within the 30-kDa protein-coding region. To resolve which of these structural features might be responsible for the decline in 30-kDa gene expression from packaged 12-RNAs, the products encoded in vitro by packaged or naked genomic and subgenomic RNAs from two strains of TMV, vulgare and a cowpea strain (Cc or Sunn-hemp mosaic virus), were compared. The results indicate that strong coat protein-RNA interactions, presumed to occur at the assembly origin, dictate the site at which translocation of 80 S ribosomes is inhibited. The implications of this conclusion for virus infection in vivo are discussed.     

The specific involvement of coat protein in tobacco mosaic virus cross protection

Nicotiana sylvestris infected by strains of tobacco mosaic virus (TMV) causing mosaic can be superinfected in the dark green leaf tissue, but not light green tissue, by necrotizing strains of TMV. The dark green tissue, however, is much less susceptible than healthy tissue, to some extent, even to unrelated viruses. The RNA of necrotizing strains of TMV was relatively more infectious than intact virus on mosaic than on healthy leaves and caused lesions in both light and dark green tissues. The same relationship was found in Nicotiana longiflora and, when the protecting strain in N. sylvestris could be used as a challenge, in Capsicum baccatum. The efficiency of superinfection by RNA was not found with viruses unrelated to TMV. When bentonite at 1 mg/ml, which is known to strip protein from TMV, was included in the inoculum of intact TMV it superinfected in the same manner as RNA. RNA of a necrotizing strain of TMV, encapsidated in brome mosaic virus protein and used as a challenge, superinfected in the same manner as RNA. When encapsidated in common TMV protein, however, it behaved as native virus. Cross protection apparently results from the prevention of uncoating of related challenge virus in light green tissue of N. sylvestris. Locally inoculated N. sylvestris leaves were insusceptible to challenge RNA or intact virus when the protecting virus was increasing. After increase ceased, RNA was more infectious than intact virus.