THE EXONUCLEASE ACTIVITY OF HSV-1 UL12 IS REQUIRED FOR IN-VIVO FUNCTION

The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline pH-dependent deoxyribonuclease termed alkaline nuclease. A recombinant UL12 knockout mutant, AN-1, is severely compromised for growth, and a nalysis of this mutant suggests that UL12 plays a role in processing c omplex DNA replication intermediates (R. Martinet, R. T Sarisky, P. C. Weber, and S. K. Weller, (1996) J. Virol. 70, 2075-2085). This proces sing step may be required for the generation of capsids that are compe tent for egress from the nucleus to the cytoplasm. In this report, we address the question of whether the AN-1 growth phenotype is due to th e loss of UL12 catalytic activity. We constructed two point mutations in a highly conserved region (motif II) of UL12 and purified wild-type and mutant enzymes from a baculovirus expression system. Both mutant proteins are stable, soluble, and competent for correct nuclear locali zation, suggesting that they have retained an intact global conformati on. Neither mutant protein, however, exhibits exonuclease activity. In order to examine the in vivo effects of these mutations, we determine d whether expression of mutant proteins from amplicon plasmids could c omplement AN-1. While the wild-type plasmid complements the growth of the null mutant, neither UL12 mutant can do so, Loss of exonuclease ac tivity therefore correlates with loss of in vivo function. (C) 1998 Ac ademic Press.