A series of frameshift and deletion mutations was created in the genome of satellite tobacco mosaic virus (STMV) by modifying full-length cDNA clones of the type strain, from which biologically active transcripts could be synthesized in vitro. Deletions and frameshift mutations in the 5' open reading frame had no effect, compared to wild-type STMV, on RNA accumulation, systemic movement, or the symptoms induced by STMV in Nicotiana tabacum co-inoculated with tobacco mild green mosaic tobamovirus (TMGMV). This implies that the protein encoded by this reading frame is not necessary for biological activity. Deletions and frameshift mutations in the coat protein open reading frame resulted in decreased accumulation of STMV RNA in N. tabacum, although these mutants were still capable of systemic movement, presumably in a nonencapsidated or free RNA form. Furthermore, the mild symptoms induced in tobacco by co-inoculations of wild-type STMV/TMGMV or infection with TMGMV alone were altered to severe systemic necrosis when plants were co-inoculated with these STMV coat protein mutants and TMGMV. Mutants within the 3' untranslated region were much less able to accumulate in TMGMV-infected plants than was wild-type STMV, and under some growth conditions did not accumulate to detectable levels.

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RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.Satellite tobacco mosaic virus (STMV) shares, with other satellite viruses and satellite RNAs, a requirement for a helper virus, in this case one of several tobamoviruses, to establish infection. Based on studies in whole tobacco plants, the helper virus is believed to provide factors required for the replication of STMV (Valverde et al., 1986(Valverde et al., , 1987(Valverde et al., , 1991. Subclones of STMV cDNA, from which biologically active RNA can be transcribed, have been created (Mirkov et al., 1990 ;Kurath et al., 1993). Interestingly, these transcripts only accumulated in tobacco plants when co-inoculated with tobacco mild green mosaic virus (TMGMV ; a California isolate previously referred to as TMV-U5 was used). In contrast, STMV RNA purified from virions accumulated systemically in plants when coinoculated with any one of several tobamoviruses (Valverde et al., 1991). Deletion and frameshift mutations have been engineered into one of the original STMV cDNA clones. Approximately 80 % of the internal coat protein (CP) coding sequence can be removed without entirely abolishing the ability of these mutants to accumulate in whole plants ; however, these CP-deletion mutants did not accumulate in every plant that was co-inoculated with mutant STMV RNA and TMGMV RNA. Viability of these STMV CP mutants was affected by the conditions under which the plants were maintained, and when they were biologically active they accumulated to approximately 100-fold lower levels than the original clone (Routh et al., 1995).To initiate an analysis of the replication of wild-type and mutant STMV, Nicotiana benthamiana protoplasts were inoculated with combinations of various tobamoviruses and STMV RNAs. The objectives of this study were to verify that replication of STMV was dependent upon factors provided by the helper virus, to determine if the specificity for TMGMV helper exhibited by cloned STMV RNA occurred at the level of replication, and to analyse the replication efficiency of STMV CP mutants which accumulated poorly in whole tobacco plants. ...

Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), two putative closteroviruses. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on this dsRNA using degenerate oligonucleotides designed to amplify an approximately 550- to 650-nucleotide fragment from the heat shock protein 70 homolog (HSP70) of the known closteroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-PCR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated from a plant infected with GLRaV-5. These clones were also sequenced. The two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate that degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detection of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR detection of multiple GLRaV serotypes. Lastly, the presence of closterovirus-like HSP70 sequences in these putative closteroviruses implies that they are indeed members of this taxonomic group.

Diagnostic methods employing the polymerase chain reaction (PCR) provide the most sensitive means currently available for detecting viruses in woody plants. A new technique has been tested that does not rely on gel electrophoresis or molecular hybridization to detect virus-specific PCR products. This colorimetric method for detection of PCR products from woody plants was demonstrated to be at least as sensitive as gel analysis. When combined with immunocapture of virions from plant sap, colorimetric detection provides a means to apply PCR technology to a large number of samples. Here, we report on the use of this technique for detection and quantitation of a walnut isolate of cherry leafroll virus (CLRV-W), citrus tristeza virus (CTV), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and tomato ringspot virus (ToRSV) in woody and herbaceous plants. For purified virus preparations, detection limits ranged from 100 pg/ml to 100 ag/ml. We also describe the colorimetric PCR detection of CTV in pooled samples.