Identification and characterization of the gene encoding the major structural protein of insect iridescent virus type 22

Abstract

The major structural protein (MSP-apparent molecular weight 49,000) of insect iridescent virus type 22 (isolated from blackflies- Simulium spp.) was resolved from disrupted, purified virus particles by SDS-PAGE and transferred to nitrocellulose by Western blotting. The portion of the blot containing the MSP was identified and excised. Tryptic peptides, generated by digestion in situ, were purified by HPLC. Three of these peptides were sequenced and an oligonucleotide gene probe was designed using one of them. A SalI clone of IV22 DNA was identified as MSP-specific by hybridization. DNA from this and an overlapping DNA clone was sequenced and a large open reading frame was positively identified as the MSP coding sequence by comparison with the tryptic peptide sequences. The molecular weight of the predicted protein product of this gene is 51,993, comparable with the apparent weight obtained by SDS-PAGE. In infected Spodoptera frugiperda (Sf) cells MSP is synthesized from 12 hr postinfection onwards. The identification of this gene and analysis of its expression opens the way to elucidating the control of late gene expression in an insect iridescent virus.