THE UL5 GENE OF HERPES-SIMPLEX VIRUS TYPE-1 - ISOLATION OF A LACZ INSERTION MUTANT AND ASSOCIATION OF THE UL5 GENE-PRODUCT WITH OTHER MEMBERS OF THE HELICASE-PRIMASE COMPLEX

The UL5 gene product is required continuously during viral DNA synthesis (L. Zhu and S. K. Weller, Virology 166:366-378, 1988) and has been shown to be a component of a three protein helicase-primase complex encoded by herpes simplex virus type 1 (J. J. Crute, T. Tsurumi, L. Zhu, S. K. Weller, P. D. Olivo, M. D. Challberg, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 86:2186-2189, 1989). The other members of the complex are viral proteins encoded by genes UL8 and UL52. In this study, we isolated a permissive cell line (L2-5) which contains the wild-type UL5 gene under the control of the strong and inducible promoter for the large subunit of herpes simplex virus type 1 ribonucleotide reductase (ICP6). An insertion mutant containing a mutation in the UL5 gene (hr99) was isolated by using the insertional mutagen ICP6::lacZ, in which the Escherichia coli lacZ gene is expressed under control of the viral ICP6 promoter. When grown on Vero cells, hr99 does not form plaques or synthesize viral DNA, although both defects are complemented efficiently on the L2-5 cells. These results confirm that the UL5 gene product is essential for viral growth and DNA replication. Furthermore, since no detectable UL5 protein is synthesized in hr99-infected cells, these cells provide a valuable control not only for the detection of the UL5 protein itself but also for the detection of protein-protein interactions with UL8 and UL52 by coimmunoprecipitation. In addition, the lacZ insertion in hr99 provides a convenient screening system for the introduction of site-specific mutations into the viral genome (L. Zhu and S. K. Weller, J. Virol. 66:469-479, 1992). Thus, hr99 is a valuable tool in the structure-function analysis of the UL5 gene.