| Herpes simplex virus type 1 (HSV-1) DNA replication is thought to progress through two distinct stages, I and II. Stage I is origin- and OBP-dependent. Stage II is origin- and OBP-independent. The events that control the switch from stage I to stage II replication are not known. However, it is likely that the switch from stage I to stage II replication involves the loss of OBP activity or OBP itself from viral origins.; A mechanism that may regulate OBP activity came to light when a naturally occurring truncated form of OBP that is in frame with and comprises the C-terminus of OBP (OBPC) was identified. Because OBPC lacks the N-terminal domains of OBP that bind UL8 and UL42, it was proposed that OBPC binds to origins, inhibiting OBP binding and blocking stage I replication. At the inception of this dissertation project, a second naturally occurring protein that is in-frame with and comprises the C-terminus of OBP (OBPC-2) was detected. Based on the size of OBPC-2, we assumed that it would retain origin-binding capabilities and hypothesized that it may also regulate OBP function.; Based on these considerations, the goal of this project was to investigate possible modes of OBP regulation by (i) determining the mechanisms of expression and roles of OBPC-1 and OBPC-2 during viral replication and (ii) defining the mechanisms of transcriptional regulation of the UL9 and UL8.5 transcripts.; I have demonstrated that OBPC-1 is a cathepsin B mediated cleavage product of OBP, whereas OBPC-2 is a product of the UL8.5 transcript. Additionally, I have demonstrated that in the absence of OBPC-1 and -2 expression, DNA levels are significantly increased at late times post infection in vitro. I also observed that mice infected with an OBPC-2 null virus displayed significantly decreased mortality rates, indicating that OBPC-2 is a mortality factor in vivo. Finally, putative UL8.5 promoter regulatory elements and a transcriptional inhibitory element in the UL9 promoter were identified. These observations indicate that OBP transcription and protein levels are regulated during infection and that OBPC-1 and OBPC-2 may act to regulate OBP function. |
