RNA 2 of soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, encodes a protein previously hypothesized to be initiated at an in-frame non-AUG codon upstream of the AUG initiation codon (nucleotide positions 334 to 336) for the 19-kDa capsid protein. Site-directed mutagenesis and in vitro transcription and translation analysis indicated that CUG (nucleotides 214 to 216) is the initiation codon for a protein with a calculated molecular mass of 25 kDa composed of a

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... ino-acid extension to the N terminus of the 19-kDa capsid protein. A stable deletion mutant, which was isolated after extensive passages of a wild-type SBWMV, contained a mixture of two deleted RNA 2's, only one of which coded for the 25-kDa protein. The amino acid sequence of the N-terminal extension was moderately conserved and the CUG initiation codon was preserved among three SBWMV isolates from Japan and the United States. This amino acid sequence conservation, as well as the retention of expression of the 25-kDa protein in the stable deletion mutant, suggests that the 25-kDa protein is functional in the life cycle of SBWMV. This is the first report of a non-AUG translation initiation in a plant RNA virus genome.