DELETION MUTANTS OF THE HERPES-SIMPLEX VIRUS TYPE-1 UL8 PROTEIN - EFFECT ON DNA-SYNTHESIS AND ABILITY TO INTERACT WITH AND INFLUENCE THE INTRACELLULAR-LOCALIZATION OF THE UL5 AND UL52 PROTEINS

The herpes simplex virus type 1 (HSV-1) helicase-primase, an essential component of the viral DNA replication machinery, is a trimeric compl ex of the virus-coded UL5, UL8, and UL52 proteins. An assembly of the UL5 and UL52 subunits retains both enzymic activities, and the UL8 pro tein has been implicated in modulating these functions, facilitating e fficient nuclear uptake of the complex and interacting with other vira l DNA replication proteins. To further our understanding of UL8, we ha ve constructed plasmids expressing mutant proteins, truncated at their N- or C-termini or lacking amino acids internally, under the control of the human cytomegalovirus major immediate-early promoter. Deletion of 23 amino acids from the N-terminus or 33 from the C-terminus abolis hed the ability of UL8 to support DNA replication in transient transfe ction assays. None of the UL8 mutants tested exhibited a strong domina nt negative phenotype in the presence of the wild-type product, althou gh some inhibition of replication was observed with mutants lacking 16 5 N-terminal or 497 C-terminal amino acids. The ability of the UL8 mut ants to facilitate efficient nuclear localization of UL52 in the prese nce of coexpressed UL5 was examined by immunofluorescence. Selected mu tants were also expressed by recombinant baculoviruses and tested for interaction with UL5 and UL52 in immunoprecipitation assays. The repli cative ability of the mutants was found to correlate with their abilit y to localize UL52 to the nucleus, but not their interaction with UL5 and UL52. This property precluded the identification oi any region of UL8 important for its presumed nuclear functions. (C) 1997 Academic Pr ess.