The coat protein genes of squash mosaic virus: cloning, sequence analysis, and expression in tobacco protoplasts

Abstract

Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified 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 2510 bp included a 2373 bp open reading frame (ORF) (encoding 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 protein 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 small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.

Structural Fingerprinting: Subgrouping of Comoviruses by Structural Studies of Red Clover Mottle Virus to 2.4-Å Resolution and Comparisons with Other Comoviruses

“…However, the extensions of this approach to other comoviruses were not particularly revealing, and no obvious discriminating criteria could be derived for subgrouping Andean potato mottle virus (APMV) (46) and several strains of squash mosaic viruses (SqMV) (15,19). The results were especially ambiguous in the case of APMV, which shares a high degree of similarity with BPMV but a comparatively low degree of similarity with CPSMV (15).…”

“…It is therefore reasonable to suggest that, as in the case of BPMV, a pentameric annular structure is not formed, supporting the previous subgrouping of CPSMV with BPMV. When the structural alignments are extended to include the sequences of the S proteins of APMV (46) and SqMV (15,19), subgrouping with BPMV is predicted in each case (Fig. 8).…”

Nucleotide sequence of a Japanese isolate of Squash mosaic virus

“…The RNA-2 of three SqMV isolates in the United States, K-SqMV, Z-SqMV [8], and H-SqMV [10], has been sequenced. K-SqMV and Z-SqMV represent two different subgroups, and display 88.1% and 89.5% identities in the nucleotide sequence of the larger and smaller CP coding regions, respectively.…”

“…Viral RNA was extracted from purified preparation of the virions by phenol/chloroform extraction, and ethanol-precipitated as previously described [10]. The RNAs, when separated by 1% agarose gel electrophoresis, formed two bands (RNA-1 and RNA-2).…”

“…The size of the RNA was considered for that expected for the RNA-1 and RNA-2 of a comovirus (data not shown). cDNA was synthesized using mixture of RNA-1 and RNA-2 and oligo (dT)-priming as previously described [10,18]. The cDNA was blunt-end ligated into the Sma I site of pUC 19 plasmids, and used to transform Escherichia coli competent cells.…”