Mapping and Expression of Southern Bean Mosaic Virus Genomic and Subgenomic RNAs

Abstract

The coat protein of the cowpea strain of southern bean mosaic sobemovirus (SBMV-C) is translated from a subgenomic RNA (sgRNA) that is synthesized in the virus-infected cell. Like the SBMV-C genomic RNA, the sgRNA has a viral protein (VPg) covalently bound to its 5' end. The mechanism(s) by which ribosomes initiate translation on the SBMV-C RNAs is not known. To begin to characterize the translation of the sgRNA it was first necessary to precisely map its 5' end. Primer extension was used to identify SBMV-C nucleotide (nt) 3241 as the transcription start site. As a control, the 5' end of the genomic RNA was also mapped. Surprisingly, the 5' terminal nt of this RNA was identified as SBMV-C nt 2. The primary structure of the 5' ends of these two RNAs is therefore expected to be VPg-ACAAAA. Precise mapping of the 5' end of the sgRNA of the bean strain of SBMV (SBMV-B) demonstrated that it has these same elements. Translation of coat protein from the SBMV-C sgRNA and p21 from the SBMV-C genomic RNA was compared using a cell-free system. The results of these experiments were consistent with translation of these proteins by a 5' end-dependent scanning mechanism rather than by internal ribosome binding.

Sobemoviruses

“…Primer extension experiments have been used to identify the precise 5Ј ends of the genomic RNAs as a start point for plus-strand synthesis. The 5Ј-terminal nucleotides of the SCPMV genomic RNA were identified as ACAAAA (36). The 5Ј ends of the genomic RNAs of two other sobemoviruses, LTSV and RYMV-CI, have been reported to be ACAAA and ACAA, respectively (114; Jeffries et al, GenBank accession no.…”

“…The 5Ј ends of the SBMV and SBMV-Ark genomic RNAs start with a similar motif, CACAAAA (58,70). At the same time, the 5Ј ends of the SCPMV and SBMV sgRNAs map to bases 3241 and 3163, respectively (36), and, based on the sequences of the SCPMV and SBMV genomes (70,112), also possess the sequence ACAAAA. These results demonstrate that the genomic RNAs and sgRNAs of sobemoviruses start with very similar primary sequences and that these are potentially important for virus replication.…”

“…In contrast, this sequence motif is not present at the 5Ј end of the CfMV genome (61,86). All sobemoviruses have a polypurine tract, including the sequence aAGgAAA (lowercase indicates less conservation of that base) just at the beginning of the genomic RNA (36).…”

P1 Protein of Cocksfoot Mottle Virus is Indispensable for the Systemic Spread of the Virus

“…The 5¢ end of viral RNA has a VPg and the 3¢ terminus lacks a poly(A) tail [2]. The genomic RNA of sobemoviruses expresses a P1 from the 5¢ ORF1 and a polyprotein (Pro-VPg-RdRp) precursor from the downstream ORF2 [3][4][5] via a leaky scanning mechanism [6].…”

Cymbidium chlorotic mosaic virus, a new sobemovirus isolated from a spring orchid (Cymbidium goeringii) in Japan

“…2a). This sequence was mapped to the stem region of a predicted stem-loop structure (data not shown) that is assumed to be important for CP subgenomic RNA transcription [9]. The fifth ORF of unknown function (ORFx), which partially overlaps with both ORF1 and ORF2a, was predicted to encode a 15.4-kDa protein (144 aa) when the translation is initiated at a non-AUG initiation site 294 GAGAUCG 300 [15].…”