Expression of the nonstructural proteins of Sindbis virus in insect cells by a baculovirus vector.
The genome of Sindbis virus encodes the polypeptides that are required for the replication and transcription of the virus RNA in infected cells. These polypeptides are translated as a polyprotein that is co- and post-translationally cleaved by an autoproteinase to give rise to four polypeptides designated nsP1, nsP2, nsP3 and nsP4. We have initiated a study of the functions of these proteins by expressing them in the Autographa californica baculovirus polyhedrin expression system. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized the four Sindbis polypeptides. We used a complementation assay which measures chloramphenicol acetyltransferase (CAT) activity to demonstrate that these proteins were biologically active. The infected cells were transfected with a Sindbis defective RNA that contains the CAT gene downstream of the promoter for the synthesis of the viral subgenomic RNA. CAT activity was found only in cells that had been infected with the recombinant baculovirus, not with wild type baculovirus, indicating that the required Sindbis nsP activities were present. Sindbis virions grew poorly in S. frugiperda cells and self-replicating Sindbis RNAs produced only very low levels of biological activity. Our results suggest that these cells are defective in their ability to replicate Sindbis RNAs and that the block is partially overcome when the Sindbis nsP mRNA is expressed under the control of the baculovirus DNA.
Buzan
J M
JM
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Schlesinger
S
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012845:Sindbis Virus / Q000235:genetics*; D014036:Togaviridae Infections / Q000235:genetics; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics*; D017361:Viral Nonstructural Proteins
D011994:Recombinant Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins