UL5, A protein required for HSV DNA synthesis: Genetic analysis, overexpression in Escherichia coli, and generation of polyclonal antibodies

The mutations in two DNA-negative is mutants of herpes simplex virus type 1 (HSV-1), tsK13 and tsM19, have been previously mapped to a 2.0-kb fragment (coordinates 0.095-0.108) at the left end of the genome (S. Weller, D. Aschman, W. Sacks, D. Coen, and P. Schaffer, 1983, Virology 130, 290-305). Sequence analysis of the HSV-1 genome has revealed the existence of two open reading frames, UL5 and UL6, within this fragment (D. McGeoch, M. Dalrymple, A. Dolan, D. McNab, L. Perry, P. Taylor, and M. Challberg,1988, J. Virol. 62, 444-453). In this paper we report fine mapping and sequence analysis of the mutations in tsK13 and tsM19 which unambiguously localize the mutations to UL5, predicted to encode a 99-kDa polypeptide. The mutation in tsK13 was shown to result in a single amino acid substitution, Pro236 to Leu, whereas tsM19 contains two substitutions, Pro236 to Ser and Ala249 to Val. Thus, both mutants are altered in Pro236. Temperature-shift experiments indicated that the UL5 gene product is required continuously during viral DNA synthesis, suggesting a direct role for the 99K protein in viral DNA synthesis. The UL5 gene product was overexpressed in Escherichia coli and used to generate polyclonal antibodies which detected proteins in HSV-1-infected cell extracts from 4 hr postinfection. Although a faint band of the predicted size (99 kDa) was observed, the majority of the immunoreactive material migrated as smaller bands which represent either proteolytic degradation during extraction or post-translational proteolytic modification of the UL5 gene product. Indirect immunofluorescence staining revealed that the UL5 gene product localizes to the nucleus in two patterns: diffuse staining throughout the nucleus and in discrete globules which appear at the periphery of the nucleus. Sequence analysis of the UL5 gene predicts that DNA 99-kDa protein contains a consensus sequence for an ATP binding site. Poss ible roles of this protein in viral synthesis are discussed.