THE PRODUCT OF THE UL12.5 GENE OF HERPES-SIMPLEX VIRUS TYPE-1 IS A CAPSID-ASSOCIATED NUCLEASE

The UL12 open reading frame of herpes simplex virus type 1 (HSV-1) enc odes a deoxyribonuclease that is frequently referred to as alkaline nu clease (AN) because of its high pH optimum. Recently, an alternate ope n reading frame designated UL12.5 was identified within the UL12 gene. UL12.5 and UL12 have the same translational stop codon, but the forme r utilizes an internal methionine codon of the latter gene to initiate translation of a 60-kDa amino terminal truncated form of AN. Since th e role of the UL12.5 protein in the HSV-1 life cycle has not Set been determined, its properties were investigated in this study. Unlike AN, which can be readily solubilized from infected cell lysates, the UL12 .5 protein was found to be a highly insoluble species, even when isola ted by high-salt detergent lysis. Since many of the structural polypep tides which constitute the HSV-I virion are similarly insoluble, a pot ential association of UL12.5 protein with virus particles was examined . By using Western blot analysis, the UL12.5 protein could be readily detected in preparations of intact virions, isolated capsid classes, a nd even capsids that had been extracted with 2 M guanidine-HCl. In con trast, AN was either missing or present at only low levels in each of these structures. Since the inherent insolubility of the UL12.5 protei n prevented its potential deoxyribonuclease activity from being assaye d in infected-cell lysates, partially purified fractions of soluble UL 12.5 protein were generated by selectively solubilizing either insolub le infected cell proteins or isolated capsid proteins with urea and re naturing them by stepwise dialysis. Initial analysis of these preparat ions revealed that they did contain an enzymatic activity that was not present in comparable fractions from cells infected with a UL12.5 nul l mutant of HSV-1. Additional biochemical characterization revealed th at UL12.5 protein was similar to AN nifh respect to pH optimum, ionic strength, and divalent cation requirements and possessed both exonucle olytic and endonucleolytic functions. The finding that the UL12.5 prot ein represents a capsid-associated form of AN which exhibits nucleolyt ic activity suggests that it may play some role in the processing of g enomic DNA during encapsidation.