FUNCTIONAL ORDER OF ASSEMBLY OF HERPES-SIMPLEX VIRUS-DNA REPLICATION PROTEINS INTO PREREPLICATIVE SITE STRUCTURES

Herpes simplex virus replicates its DNA within nuclear structures call ed replication compartments. In contrast, in cells in which viral DNA replication is inhibited, viral replication proteins localize to punct ate structures called prereplicative sites. We have utilized viruses i ndividually mutated in each of the seven essential replication genes t o assess the function of each replication protein in the assembly of t hese proteins into prereplicative sites. We observed that four replica tion proteins, UL5, UL8, UL52, and UL9, are necessary for the localiza tion of ICP8 (UL29) to prereplicative sites under natural infection co nditions. Likewise, four of the seven viral DNA replication proteins, UL5, UL52, UL9, and ICP8, are necessary for the localization of UL8 to these sites. In contrast, all six of the other replication proteins a re necessary for efficient localization of the viral DNA polymerase to prereplicative sites. On the basis of these results, we present a mod el for prereplicative site formation in infected cells in which the he licase-primase components (UL5, UL8, and UL52), the origin-binding pro tein (UL9), and the viral single-stranded DNA-binding protein (ICP8) a ssemble together to initiate the process. This is followed by the recr uitment of the viral polymerase into the structures, a step facilitate d by the polymerase accessory protein, UL42. Host cell factors can app arently substitute for some of these viral proteins under certain cond itions, because the viral protein requirements for prereplicative site formation are reduced in transfected cells and in infected cells trea ted with drugs that inhibit DNA synthesis.