Temperature-sensitive origin-binding protein as a tool for investigations of herpes simplex virus activities in vivo.

While it is fairly clear that herpes simplex virus (HSV) DNA replication requires at least seven virus-encoded proteins in concert with various host cell factors, the mode of this process in infected cells is still poorly understood. Using HSV-1 mutants bearing temperature-sensitive (ts) lesions in the UL9 gene, we previously found that the origin-binding protein (OBP), a product of the UL9 gene, is only needed in the first 6 hours post-infection. As this finding was just a simple support for the hypothesis of a biphasic replication mode, we became convinced through these earlier studies that the mutants tsR and tsS might represent suitable tools for more accurate investigations in vivo. However, prior to engaging in highly sophisticated research projects, knowledge of the biochemical features of the mutated versions of OBP appeared to be essential. The results of our present study demonstrate that (i) tsR is most appropriate for cell biological studies, where only immediate early and early HSV gene products are being expressed without the concomital viral DNA replication, and (ii) tsS is a prime candidate for the analysis of HSV DNA replication processes because of its reversibly thermosensitive OBP-ATPase, which allows one to switch on the initiation of DNA synthesis precisely.

Genome replication and progeny virion production of herpes simplex virus type 1 mutants with temperature-sensitive lesions in the origin-binding protein.

Genome replication of herpes simplex viruses (HSV) in cultured cells is thought to be started by the action of the virus-encoded origin-binding protein (OBP). In experiments using two HSV-1 mutants with temperature-sensitive lesions in the helicase domain of OBP, we demonstrated that this function is essential during the first 6 hours of the lytic cycle. Once DNA synthesis has started, this function is no longer required, suggesting that origin-driven initiation of viral DNA replication is a single event rather than a continuous process.