Baculovirus polyhedrin promoter-directed expression of rubella virus envelope glycoproteins, E1 and E2, in Spodoptera frugiperda cells.
To study the capability of Spodoptera frugiperda (fall armyworm; Sf9) cells to synthesize and process mature rubella virus (RV) proteins, a cDNA encoding the structural envelope glycoproteins, E1 (58 kDa) and E2 (42-47 kDa) were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and expressed during infection under the transcriptional regulation of the polyhedrin gene promoter. By immunoblot analysis with antibodies directed against purified RV, the individual proteins E1 and E2, and human convalescent serum, a polyprotein precursor migrating with an apparent molecular weight of 90-95 kDa was identified in Sf9 cells infected with the recombinant baculovirus, Ac701-RVE. In addition, two proteins migrating somewhat faster than authentic viral E1 and E2 were resolved. Pulse-chase labeling experiments in the absence and presence of tunicamycin, as well as treatment of the recombinant proteins with endo-beta-N-acetyl-D-glucosaminidase H indicated that the recombinant proteins are glycosylated and that the E1 and E2 apoproteins, respectively, were similar in size as compared to their in vitro synthesized counterparts. The recombinant protein products were further detected by some monoclonal antibodies directed against RV. The results presented here indicate that a polyprotein containing the envelope glycoproteins of RV is expressed and proteolytically cleaved in lepidopteran insect cells to form two proteins which resemble authentic E1 and E2. The baculovirus may therefore be suitable for the abundant expression of RV antigen.
Oker-Blom
C
C
Department of Entomology, Texas, A&M University, College Station.
Pettersson
R F
RF
Summers
M D
MD
D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors; D006596:Hexosaminidases / Q000494:pharmacology; D007304:Insect Viruses / Q000235:genetics*; D008970:Molecular Weight; D009036:Moths / Q000166:cytology; D000077183:Occlusion Body Matrix Proteins; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012412:Rubella virus / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D000077183:Occlusion Body Matrix Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; D006596:Hexosaminidases