HERPES-SIMPLEX VIRUS TYPE-1 PREREPLICATIVE SITES ARE A HETEROGENEOUS POPULATION - ONLY A SUBSET ARE LIKELY TO BE PRECURSORS TO REPLICATION COMPARTMENTS

When herpes simplex virus type 1 (HSV-1) DNA replication is blocked by viral polymerase inhibitors, such as phosphonoacetic acid (PAA) or ac yclovir (ACV), UL29 (ICP8) localizes to numerous punctate nuclear foci which are called prereplicative sites. Since this pattern can form in cells infected with mutants which are defective in UL5, UL8, UL9, or UL52 in the presence of polymerase inhibitors (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996; L. M. Liptak, S. L. Uprichard, and D. M. Knipe, J. Virol. 70:1759-1767, 1996), we previously proposed that it is unlikely that these numerous UL29 foci actually represent a functional subassembly of viral replication proteins that could lead to the formation of replication compartments (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996). In this paper, we have investi gated the requirement for formation of the prereplicative site pattern by using double mutants of HSV. From the analysis of mutants lacking both UL5 and UL9, we conclude that neither viral helicase is required for the prereplicative site pattern to form as long as a polymerase in hibitor is present. From the analysis of mutants defective in both UL3 0 and UL5, we suggest that the prereplicative site pattern can form un der conditions in which viral and/or cellular polymerases are inhibite d. Furthermore, reexamination of the UL29 staining pattern in cells in fected with wild-type virus in the presence of PAA reveals that at lea st two different UL29 staining patterns can be detected in these cells . One population of cells contains numerous (greater than 20) punctate UL29 foci which are sites of cellular DNA synthesis. In another popul ation of cells, fewer punctate foci (less than 15) are detected, and t hese structures do not colocalize with sites of cellular DNA synthesis . Instead, they colocalize with PML, a component of nuclear matrix str uctures known as ND10. We propose that ND10-associated UL29 sites repr esent domains at which replication compartments form.