Overexpression and assembly of the herpes simplex virus type 1 helicase-primase in insect cells.
Herpes simplex virus type 1 (HSV-1) encodes a helicase-primase that consists of three polypeptides encoded by the UL5, UL8, and UL52 genes (Crute, J.J., Tsurumi, T., Zhu, L., Weller, S.K., Olivo, P.D., Challberg, M.D., Mocarski, E.S., and Lehman, I.R. (1989) Proc. Natl. Acad, Sci, U.S.A. 86, 2186-2189). To obtain sufficient quantities of the enzyme for study, we have overexpressed the three genes using the baculovirus expression system. We find that the fully active enzyme can be assembled in vivo by triply infecting Spodoptera frugiperda SF9 cells with a baculovirus recombinant for each gene. The recombinant enzyme which we have purified to near homogeneity from the insect cells has a molecular weight of 270,000 and is composed of the three polypeptides encoded by the UL5, UL8, and UL52 genes. The enzyme possesses DNA-dependent ATPase, DNA-dependent GTPase, DNA helicase, and DNA primase activities that are essentially identical to the enzyme isolated from HSV-1-infected cells.
Dodson
M S
MS
Department of Biochemistry, Beckman Center, Stanford University, California 94305-5307.
Crute
J J
JJ
Bruckner
R C
RC
Lehman
I R
IR
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D015870:Gene Expression; D005814:Genes, Viral; D015151:Immunoblotting; D007304:Insect Viruses / Q000235:genetics; D007313:Insecta; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics*
D011994:Recombinant Proteins; D015678:Viral Structural Proteins