Expression of Sindbis virus 26S cDNA in Spodoptera frugiperda (Sf9) cells, using a baculovirus expression vector.
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 E1 protein, of 56 kDa) was inserted into the genome of Autographa 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 E1), 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-beta-N-acetylglucosaminidase H (endo-H) treatment of the recombinant glycoproteins, and by N-terminal sequence analysis of the E1 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.
Oker-Blom
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C
Department of Entomology, Texas A & M University, College Station 77843-2475.
Summers
M D
MD
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006596:Hexosaminidases / Q000494:pharmacology; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics*; D014415:Tunicamycin / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D014415:Tunicamycin; D004247:DNA; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase