Title: Mechanism of expression and functional analysis of herpes simplex virus type-1 proteins OBPC-1 and OBPC-2
Description: 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.