Characterization of Deletion and Frameshift Mutants of Satellite Tobacco Mosaic Virus

Abstract

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.

Replication of tobacco mosaic virus RNA

“…The 1059-nucleotide STMV RNA contains two open reading frames, one for a 6.6-kDa protein of unknown function and the other for its 17.5-kDa coat protein. Neither of these proteins is required for replication of STMV (Routh et al 1995(Routh et al , 1997, which relies completely on replication proteins of its tobamovirus helper. The natural helper for STMV is tobacco mild green mosaic virus (TMGMV), although other tobamoviruses, including TMV-U1, can act as less e¤cient helpers.…”

In Vitro- and In Vivo-Generated Defective RNAs of Satellite Panicum Mosaic Virus Define cis -Acting RNA Elements Required for Replication and Movement

“…Furthermore, the signals for replication and movement are embedded in the 5Ј-proximal 16 nt, nt 62 to 110 in the 5Ј end, and the entire 3Ј UTR plus the 73 nt upstream of the CP stop codon on SPMV RNA. The delineation of cis-acting elements in three satellite viruses, STNV (1), STMV (16,17), and SPMV, supports the implication that the crucial signals for the initial replication cycle and systemic movement are clustered within the UTRs of the satellite viral RNA genomes. An interesting question that has not yet been addressed is why SPMV and other satellite viruses retain their respective CP genes even though they are dispensable for the essential life cycles of satellite viruses.…”

“…2 and 3). UTRs (1,12,16,17). For example, the cis-acting elements necessary for the replication of STNV are embedded in three hairpin-like structures on the 5Ј UTR and in two discrete regions identified as the 3Ј-proximal terminal sequence and the sequence immediately adjacent to the CP ORF in the 3Ј UTR (1).…”

“…For example, the cis-acting elements necessary for the replication of STNV are embedded in three hairpin-like structures on the 5Ј UTR and in two discrete regions identified as the 3Ј-proximal terminal sequence and the sequence immediately adjacent to the CP ORF in the 3Ј UTR (1). Likewise, a replication competency assay with STMV deletion mutants revealed that the 3Ј UTR sequence immediately downstream of the CP ORF was critical for STMV accumulation (16,17). In contrast, the CP genes of both STNV and STMV were dispensable for accumulation and movement (1,16,17).…”

“…Likewise, a replication competency assay with STMV deletion mutants revealed that the 3Ј UTR sequence immediately downstream of the CP ORF was critical for STMV accumulation (16,17). In contrast, the CP genes of both STNV and STMV were dispensable for accumulation and movement (1,16,17).…”