The 105-kDa polyprotein of southern bean mosaic virus is translated by scanning ribosomes

The cowpea strain of southern bean mosaic virus (SBMV-C) is a positive-sense RNA virus. Three open reading frames (ORF-1, ORF2, and ORF3) are expressed from the genomic RNA. The ORF1 and ORF2 initiation codons are located at nucleotide (nt) positions 49 and 570, respectively. ORF1 is expressed by a 5' end-dependent scanning mechanism, but it is not known how ribosomes gain access to the ORF2 initiation codon. In experiments described here, it was demonstrated that the translation of ORF2 was sensitive to cap analog in a cell-free extract. In vitro and in vivo studies showed that the addition of one or more AUG codons between the 5' end of the SBMV-C RNA and the ORF2 initiation codon reduced ORF2 expression and that elimination of the ORF1 initiation codon increased ORF2 expression. Altering the sequence context of the ORF1 initiation codon to one more favorable for translation initiation also reduced ORF2 expression in vivo. Nucleotide deletions and insertions between SBMV-C nt 218-520 did not abolish ORF2 expression. In most cases, these mutations resulted in reduced expression of both ORF1 and ORF2. These results are consistent with translation of ORF2 by leaky scanning.