ASSEMBLY OF COMPLETE, FUNCTIONALLY ACTIVE HERPES-SIMPLEX VIRUS-DNA REPLICATION COMPARTMENTS AND RECRUITMENT OF ASSOCIATED VIRAL AND CELLULAR PROTEINS IN TRANSIENT COTRANSFECTION ASSAYS

Early during the herpes simplex virus (HSV) lytic cycle or in the pres ence of DNA synthesis inhibitors, core viral replication machinery pro teins accumulate in intranuclear speckled punctate prereplicative foci , some of which colocalize with numerous sites of host cellular DNA sy nthesis initiation known as replisomes. At later times, in the absence of inhibitors, several globular or large irregularly shaped replicati on compartments are formed; these compartments also contain progeny vi ral DNA and incorporate the IE175(ICP4) transcription factor together with several cellular proteins involved in DNA replication and repair, In this study, we demonstrate that several forms of both prereplicati on foci and active viral replication compartments that display an appe arance similar to that of the compartments in HSV-infected cells can b e successfully assembled in transient assays in DNA-transfected cells receiving genes encoding all seven essential HSV replication fork prot eins together with oriS target plasmid DNA. Furthermore, bromodeoxyuri dine (BrdU)-pulse-labeled DNA synthesis initiation sites colocalized w ith the HSV single-stranded DNA-binding protein (SSB) in these replica tion compartments, implying that active viral DNA replication may be o ccurring. The assembly of complete HSV replication compartments and in corporation of BrdU were both abolished by treatment with phosphonoace tic acid (PAA) and by omission of any one of the seven viral replicati on proteins, UL5, UL8, UL9, UL42, UL52, SSB, and Pol, that are essenti al for viral DNA replication. Consistent with the fact that hath HSV I E175 and IE63(ICP27) localize within replication compartments in HSV-i nfected cells, the assembled HSV replication compartments were also ab le to recruit both of these essential regulatory proteins. Blacking vi ral DNA synthesis with PAA, but not omission of oriS, prevented the as sociation of IE175 with prereplication structures, The assembled HSV r eplication compartments also redistributed cotransfected cellular p53 into the viral replication compartments, However, the other two HSV im mediate-early nuclear proteins IE110(ICP0)and IE68(ICP22) did not ente r the replication compartments in either infected or transfected cells .