THE HERPES-SIMPLEX VIRUS TYPE-1 CLEAVAGE PACKAGING PROTEIN, UL32, IS INVOLVED IN EFFICIENT LOCALIZATION OF CAPSIDS TO REPLICATION COMPARTMENTS/

Six genes, including UL32, have been implicated in the cleavage and pa ckaging of herpesvirus DNA into preassembled capsids. We have isolated a UL32 insertion mutant which is capable of near-wild-type levels of viral DNA synthesis; however, the mutant virus is unable to cleave and package viral DNA, consistent with the phenotype of a previously isol ated temperature-sensitive herpes simplex virus type 1 mutant, tsN2O ( P. A. Schaffer, G. M. Aron, N. Biswal, and M. Benyesh-Melnick, Virolog y 52:57-71, 1973). A polyclonal antibody which recognizes UL32 was pre viously used by Chang et al, (Y. E. Chang, A. P. Poon, and B. Roizman, J. Virol. 70:3938-3946, 1996) to demonstrate that UL32 accumulates pr edominantly in the cytoplasm of infected cells, In this report, a func tional epitope-tagged version of UL32 showed that while UL32 is predom inantly cytoplasmic, some nuclear staining which colocalizes with the major DNA binding protein (ICPS, UL29) in replication compartments can be detected. We have also used a monoclonal antibody (5C) specific fo r the hexon form of major capsid protein VP5 to study the distribution of capsids during infection. In cells infected with wild-type KOS (6 and 8 h postinfection), 5C staining patterns indicate that capsids are present in nuclei within replication compartments. These results sugg est that cleavage and packaging occur in replication compartments at l east at 6 and 8 h postinfection, Cells infected with the UL32 mutant e xhibit a hexon staining pattern which is more diffusely distributed th roughout the nucleus and which is not restricted to replication compar tments. We propose that UL32 may play a role in ''bringing'' preassemb led capsids to the sites of DNA packaging and that the failure to loca lize to replication compartments may explain the cleavage/packaging de fect exhibited by this mutant, These results suggest that the UL32 pro tein is required at a step distinct from those at which other cleavage and packaging proteins are required and may be involved in the correc t localization of capsids within infected cells.