Analysis of in vitro activities of herpes simplex virus type 1 UL42 mutantproteins: Correlation with in vivo function

The DNA polymerase (pol) catalytic subunit of herpes simplex virus type 1, encoded by UL30, and its accessory factor, UL42 protein, are both essential for the replication of the virus. Because the stable interaction between U L42 and pol renders the pol fully processive for replicative DNA synthesis, disruption of this interaction represents a potential goal in the developm ent of novel antiviral compounds. To better compare the effects of mutation s in UL42 protein on its known in vitro functions. mutations were expressed as glutathione-S-transferase (GST)-fusions and the fusion proteins used in affinity chromatography. In this report, we demonstrate the relationship b etween the abilities of mutant UL42 fusion proteins to bind pol and to stim ulate pol activity in vitro, and the abilities of nonfusion mutant proteins to function in viral replication. The pol stimulation assay using GST fusi on proteins was found to be a more accurate and sensitive measure of the ab ility of the UL42 protein to function in vitro than the pol binding assay u sing the fusion proteins linked to a solid matrix. We also found an excelle nt correlation between the ability of purified GST fusion proteins to stimu late pol activity in vitro and the ability of full-length nonfusion UL42 mu tant genes to support DNA replication in infected cells. Our results demons trate that two noncontiguous stretches of amino acids, from 137 to 142 and from 274 to 282, are essential for UL42 function in vivo and in vitro, Alth ough mutant d241-261 exhibited close to wild-type abilities to stimulate po l activity in vitro, it was not capable of complementing the replication of a UL42 null mutant virus. The region of UL42 protein within or close to 24 1-261 may serve to hinge the essential regions within the N- and C-terminal portions of the protein which are thought to interdigitate. It is hypothes ized that reduction in the length of the hinge region could alter the abili ty of UL42, and/or its complex with pol, to function with one or more of th e other proteins present in the DNA replisome within infected cells, (C) 20 00 Academic Press.