STRUCTURE-FUNCTION ANALYSIS OF THE HERPES-SIMPLEX VIRUS TYPE-1 UL12 GENE - CORRELATION OF DEOXYRIBONUCLEASE ACTIVITY IN-VITRO WITH REPLICATION FUNCTION

Although the product of the UL12 gene of herpes simplex virus type 1 ( HSV-1) has been shown to possess both exonuclease and endonuclease act ivities in vitro, and deletion of most of the gene within the viral ge nome results in inefficient production and maturation of infectious vi rions, the function of the deoxyribonuclease (DNase) activity per se i n virus replication remains unclear. In order to correlate the in vitr o and in vivo activities of the protein encoded by UL12, mutant protei ns were tested for nuclease activity in vitro by a novel hypersensitiv ity cleavage assay and for their ability to complement the replication of a DNase null mutant, AN-1. Rabbit reticulocyte lysates programmed with wild-type UL12 RNA cleaved at the same sites cleaved by purified HSV-1 DNase, but distinct from those cleaved by DNase I or micrococcal nuclease. All mutants which lacked DNase activity in vitro also faile d to complement the replication of AN-1 in nonpermissive cells. Likewi se, all mutants which contained HSV-1 DNase activity, as detected by t he hypersensitivity cleavage assay, were capable of complementing the replication of the DNase null mutant, though to varying extents. Of pa rticular note was the d1-126 mutant protein, which, despite having the same specific activity as the wild-type enzyme in vitro, complemented the replication of AN-1 significantly less than the wild-type protein . The results suggest that DNase activity per se is required for effic ient replication of HSV-1 in vivo. However, residues, including the N- terminal 126 amino acids, which are dispensable for enzymatic activity in vitro may facilitate the accessibility or activity of the protein in vivo. (C) 1998 Academic Press.