ABSTRACT
We constructed recombinant viruses based on the herpes simplex virus type 1 mutant tsK which individually were able to express the products of four viral DNA replication genes (UL5, UL8, UL9 and UL52) in the absence of any of the other proteins required for viral DNA synthesis. These viruses were used in immunofluorescence experiments to investigate the cellular localization of the four replication proteins expressed. The results demonstrated that all three components of the viral helicase-primase complex (UL5, UL8 and UL52 proteins) must be co-expressed to allow their efficient localization to the nucleus. Since the UL5 and UL52 proteins together form a complex which is enzymatically indistinguishable from a complex formed from all three proteins, a possible role of the UL8 protein may be in facilitating nuclear uptake. The UL9 protein (origin-binding protein) efficiently entered the cell nucleus when expressed alone. Both UL9 protein and the tripartite helicase-primase complex exhibited patterns of fluorescence which resembled the 'pre-replicative sites' described previously.
Subject(s)
DNA Helicases/isolation & purification , DNA-Binding Proteins/isolation & purification , RNA Nucleotidyltransferases/isolation & purification , Simplexvirus/chemistry , Viral Proteins/isolation & purification , Cell Nucleus/microbiology , DNA Helicases/genetics , DNA Primase , DNA Replication , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique , Gene Expression , Protein Biosynthesis , RNA Nucleotidyltransferases/genetics , Recombination, Genetic , Viral Proteins/genetics , Virus ReplicationABSTRACT
The herpes simplex virus type 1 helicase-primase complex consists of the products of the UL5, UL8 and UL52 genes. We have expressed these proteins in insect cells using baculovirus vectors and studied the requirements for enzymatic activities associated with the DNA unwinding function of the complex. In agreement with a recent report (Dodson, M.S., Crute, J.J., Bruckner, R.C. and Lehman, I.R. 1989, J. Biol. Chem. 264, 20835-20838) we find that DNA-dependent ATPase and DNA helicase activities are assembled in vivo in insect cells triply infected with viruses expressing the UL5, UL8 and UL52 proteins. Moreover, these activities were also detected in cells in which only the UL5 and UL52 products were expressed indicating that the presence of the UL8 protein is essential for neither the ATPase nor helicase activity of the complex.
Subject(s)
Adenosine Triphosphatases/metabolism , DNA Helicases/metabolism , Multienzyme Complexes , RNA Nucleotidyltransferases/metabolism , Simplexvirus/enzymology , Viral Proteins/metabolism , Animals , Cells, Cultured , Chromatography , DNA/metabolism , DNA Primase , Gene Expression , Genes, Viral , Insecta , Simplexvirus/genetics , Viral Proteins/geneticsABSTRACT
The binding of a herpes simplex virus type 1 (HSV-1) encoded polypeptide to a viral origin of DNA replication has been studied by using a gel retardation assay. Incubation of nuclear extract from HSV-1 infected cells with a labelled origin-containing fragment resulted in the formation of a specific retarded complex, the migration of which was further reduced in the presence of an antibody reactive with the UL9 gene product. Introduction of an additional copy of the UL9 gene, under the control of an immediate early (IE) promoter, conferred the ability to express origin binding activity at the non-permissive temperature upon an HSV-1 ts mutant blocked at the IE stage of infection. Endogenous or exogenous proteolytic activity revealed the presence of a relatively protease-resistant domain which retained sequence-specific DNA binding activity. The C-terminal 317 amino acids of the UL9 gene expressed as a fusion protein in Escherichia coli also bound to the origin. Our results demonstrate that the UL9 gene product binds to a viral origin and that sequence specific recognition and binding are specified by the C-terminal 37% of the polypeptide.
