Expression and characterization of poliovirus proteins 3BVPg, 3Cpro, and 3Dpol in recombinant baculovirus-infected Spodoptera frugiperda cells.
As an initial step toward investigating the roles of poliovirus proteins in viral RNA replication, a baculovirus expression system was used to produce poliovirus proteins from the P3 region. Spodoptera frugiperda (Sf9) cells were infected with a recombinant baculovirus, vETL-PoV3A*BCD, which contains cDNA coding for poliovirus proteins 3D, 3C, 3B, and a portion of 3A protein sequence. Immunofluorescence microscopy revealed that the majority of 3D (polymerase) was in the cytoplasm of recombinant baculovirus-infected Sf9 cells. In the same cells, the 3C (protease) and 3B (VPg) proteins appeared to be located in distinct subcellular regions, possibly membrane structures, suggesting that the expressed polyprotein was cleaved to generate mature proteins. Processing of the polypeptide was confirmed by immunoblot analysis which demonstrated that 3Cpro sequences were active in cleavage of the polyproteins 3A*BCD and 3CD. Over 95% of the 3D sequences accumulated in the form of mature 3Dpol, with only low levels of 3CD remaining. The majority of 3Dpol remained in the supernatant after low speed centrifugation of sonicated cells. The 3Dpol had RNA-dependent RNA polymerase activity as measured by elongation of an oligo(U) primer using a poly(A) template. The protein 3CDpro was active in cleaving P1 protein. The yield and activities of the poliovirus proteins expressed will facilitate future biochemical studies.
Neufeld
K L
KL
Department of Cellular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Richards
O C
OC
Ehrenfeld
E
E
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D015870:Gene Expression; D009036:Moths; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
D014764:Viral Proteins