THE HUMAN CYTOMEGALOVIRUS UL98 GENE ENCODES THE CONSERVED HERPESVIRUSALKALINE NUCLEASE

The human cytomegalovirus (HCMV) UL98 gene is predicted to encode a ho mologue of the conserved herpesvirus alkaline nuclease, To determine i f the HCMV UL98 gene product is functionally homologous to other herpe svirus alkaline nucleases, the HCMV UL98 protein was purified and its activity characterized in vitro. Extracts of HCMV-infected cells were fractionated using Q Sepharose, phosphocellulose and native DNA cellul ose chromatography. UL98 immunoreactivity copurified with alkaline pH- dependent nuclease activity. The purified protein migrated at its pred icted size of approximately 65 kDa in denaturing polyacrylamide gels, and displayed nuclease activity in an activity gel assay, Enzyme activ ity was characterized by a high pH optimum, an absolute requirement fo r divalent cation, salt sensitivity, and 5' to 3' exonuclease activity . DNA digestion resulted in 5' monophosphoryl mono-and oligodeoxyribon ucleotides. Kinetic analyses revealed a turnover rate of greater than 200 per min, and similar apparent affinity and rate constants on singl e-and double-stranded DNA. These results indicate that a functional al kaline nuclease activity is conserved among distant members of the her pesvirus family, and are consistent with a highly conserved role in th e virus life cycle.