THE COAT PROTEIN GENES OF SQUASH MOSAIC-VIRUS - CLONING, SEQUENCE-ANALYSIS, AND EXPRESSION IN TOBACCO PROTOPLASTS

Complementary DNA of the middle-component RNA of the melon strain of s quash mosaic comovirus (SqMV) was cloned. Clones containing the coat p rotein genes were identified by hybridization with a degenerate oligon ucleotide synthesized according to the amino acid sequence of a purifi ed peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 25 10 bp included a 2373 bp open reading frame (ORF) (enco ding 791 amino acids), a 123 bp 3'-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protei n revealed its presence at the 3' end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and sma ll coat proteins from each other. A putative location of the N-termina l proteolytic cleavage site of the 42 k coat protein (Q/N) was predict ed by comparisons with the corresponding coat proteins of cowpea mosai c virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little si milarity, their encoded coat proteins shared about 47% identity. The i dentity of the encoded 42 k and 22 k peptides was confirmed by enginee ring the respective gene regions for expression followed by transfer i nto tobacco protoplasts using the polyethylene glycol method. Both SqM V coat proteins were expressed in vivo as determined by their reactivi ty to SqMV coat protein specific antibodies.