1. Defining the Role of the Host Chaperone Machinery During HSV-1 Infection. April D. Burch and Sandra K. Weller. Many viruses and bacteriophage utilize chaperone systems for DNA replication and viral morphogenesis. We have previously shown that in the HSV-1-infected cell nucleus, foci enriched in the Hsp70/Hsp40 chaperone machinery are formed adjacent to viral replication compartments (Burch, A.D. and Weller, S.K. 2004. Journal of Virology. 78(13):7175-7185). Since the Hsp90 chaperone machinery is known to engage the Hsp70/Hsp40 system in eukaryotes, the subcellular localization of Hsp90 in HSV-1-infected cell was analyzed. Hsp90 is found within viral replication compartments as well as in the Hsp70/Hsp40-enriched foci. Geldanamycin, an inhibitor of Hsp90, results in decreased HSV-1 yields and blocks viral DNA synthesis. Furthermore, we have found that the viral DNA polymerase is mislocalized to the cytoplasm in both infected and transfected cells in the presence of Geldanamycin. These data identify the HSV-1 polymerase as a putative client protein of the Hsp90 chaperone system. Perturbations in this association appear to result in aberrant folding and/or intracellular localization of the viral polymerase.

2. Roles of the Host Cell Response to DNA Damage During HSV-1 Infection . Dianna Wilkinson and Sandra K. Weller. In response to agents that cause DNA damage, mammalian cells activate signal transduction pathways that slow cell cycle progression and repair the damaged DNA. If the damage is irreparable, cells are eliminated through the induction of apoptosis. Defects in this stress response can compromise genomic stability resulting in accumulation of mutations and transformation to malignancy. One of the early responders to DNA damage is replication protein A (RPA), a heterotrimeric ssDNA-binding protein that plays key roles in DNA replication as well as in the recognition, signaling and repair of damaged DNA. Under DNA-damaging conditions, stretches of ssDNA arise at DNA lesions to which RPA binds and the 32kDa subunit of RPA (RPA32) becomes hyperphosphorylated. Thus the hyperphosphorylation of RPA and its presence at DNA lesions serves as a reliable cellular marker for DNA damage. We have recently reported that in the presence of the viral polymerase inhibitor, phosphonoacetic acid (PAA), herpes simplex virus type I (HSV-1) induces the hyperphosphorylation of RPA32. This DNA damage response appears to be specific to the inhibition of the viral polymerase since the hyperphosphorylation of RPA32 was not observed during productive infection or during infection with a polymerase null virus. We initiated the present study to further define this host stress response to HSV-1 infection. Here, we report that infection in the presence of polymerase inhibitors triggers a cellular response to DNA damage as demonstrated by the induction of the hyperphosphorylation of RPA and its accumulation at DNA lesions within a subpopulation of infected cells. This DNA damage response occurs only in the presence of an inhibited but otherwise functional HSV-1 polymerase and also requires the polymerase accessory protein and viral ssDNA-binding protein, but not other viral proteins essential for HSV-1 replication. Finally, this DNA damage response was not observed during productive infection, indicating that active viral replication does not trigger this potentially detrimental stress response. The ability to avoid triggering such a signal may be part of a global mechanism adopted by HSV-1 to prevent catastrophic events such as replication stress at viral forks and apoptosis.