Expression of Sindbis virus 26S cDNA in Spodoptera frugiperda (Sf9) cells, using a baculovirus expression vector

Abstract

To study protein processing in an insect Spodoptera frugiperda (fall armyworm; Sf9) cell line, a 26S cDNA encoding the sequence of Sindbis virus structural proteins (capsid protein, of 30 kilodaltons [kDa]; p62 [the precursor of E3 and E2], of 62 kDa; a 6-kDa peptide; and the El protein, of 56 kDa) was inserted into the genome ofAutographa californica nuclear polyhedrosis virus (AcNPV) adjacent to the polyhedrin promoter. By immunoblot analysis with antisera directed against whole Sindbis virus and the individual structural proteins (capsid, E2, and El), we have shown that polypeptides similar in size and antigenicity to those synthesized in Sindbis virus-infected BHK cells are expressed in Sf9 cells infected with the recombinant baculovirus Ac373-SV26. By pulse-chase labeling in the presence or absence of tunicamycin, by endo-P-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the El envelope glycoprotein, we have further shown that the 26S transcription translation unit of Sindbis virus, although normally encoded by nonnuclear RNA, is expressed and proteolytically cleaved similarly, if not identically, in Sf9 cells as compared with BHK cells when a baculovirus expression vector is used.

Strain Identification of Spodoptera Frugiperda (Lepidoptera: Noctuidae) Insects and Cell Line: PCR-RFLP of Cytochrome Oxidase C Subunit I Gene

“…Sf9 cells are a subclone of IPLB-SF-21 cells (Vaughn et al 1977) originally cultured from pupal S. frugiperda tissue used in expression vector systems (Luckow & Summers 1988aand 1988b, Oker-Blom & Summers 1989. These cells are also used to study viral infection for the development of highly specific biological pesticides (Danyluk & Maruniak 1987, Sieburth & Maruniak 1988.…”