title: Analysis of in vitro activities of herpes simplex virus type 1 UL42 mutant proteins: correlation with in vivo function.
abstract: 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 UL42 and pol renders the pol fully processive for replicative DNA synthesis, disruption of this interaction represents a potential goal in the development of novel antiviral compounds. To better compare the effects of mutations 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 between the abilities of mutant UL42 fusion proteins to bind pol and to stimulate pol activity in vitro, and the abilities of nonfusion mutant proteins to function in viral replication. The pol stimulation assay using GST fusion proteins was found to be a more accurate and sensitive measure of the ability of the UL42 protein to function in vitro than the pol binding assay using the fusion proteins linked to a solid matrix. We also found an excellent correlation between the ability of purified GST fusion proteins to stimulate pol activity in vitro and the ability of full-length nonfusion UL42 mutant genes to support DNA replication in infected cells. Our results demonstrate 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. Although mutant d241-261 exhibited close to wild-type abilities to stimulate pol 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 241-261 may serve to hinge the essential regions within the N- and C-terminal portions of the protein which are thought to interdigitate. It is hypothesized that reduction in the length of the hinge region could alter the ability of UL42, and/or its complex with pol, to function with one or more of the other proteins present in the DNA replisome within infected cells.
authors.affiliation: Program in Molecular, Cellular, and Developmental Biology, Comprehensive Cancer Center, 333 West Tenth Avenue, Columbus, Ohio 43210, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Thornton
authors.forename: M
authors.initials: M
authors.lastname: Chaudhuri
authors.forename: S J
authors.initials: SJ
authors.lastname: Monahan
authors.forename: L A
authors.initials: LA
authors.lastname: Grinstead
authors.forename: D S
authors.initials: DS
authors.lastname: Parris
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D006224:Cricetinae; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005090:Exodeoxyribonucleases; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005982:Glutathione Transferase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus