Nuclear Sensing of Herpesviral DNA - Project Summary/Abstract Nucleoside analogs such as acyclovir and other drugs are available to treat HSV infections, but viral re- sistance to the drugs can arise in certain situations, and genital herpes transmission is only partially prevented by current drugs. Therefore, additional antiviral strategies are needed. These studies will identify potential new antiviral strategies and generate new HSV gene therapy vectors and understanding of their mechanism of action. Nuclear DNA viruses face various host responses that try to block the infection when their DNA genomes enter the nucleus, and they have evolved various strategies for evading or neutralizing these host restrictions. The host responses include epigenetic silencing, DNA damage responses (DDR), and innate responses. The her- pesviruses have no chromatin on their genome in the virion, and the host cell recognizes the viral DNA as “for- eign,” loads heterochromatin on the incoming viral genome, initiates DDR, and induces innate signaling. Viral host factors that sense and respond to foreign DNA include both restriction factors and DDR factors. We have Identified two new host factors, IFI16 and TRIM22, that sense input viral DNA and contribute to viral silencing. We have also shown that Mre11, a DNA sensing factor for a DDR pathway restricts HSV-1 replication, connect- ing a DDR component to DNA sensing and silencing. In this application we investigate three new areas of host sensing of DNA virus infection: 1) the mechanisms by which IFN-induced levels of IFI16 can restrict wild-type HSV-1 replication; 2) blebbing of nuclear DNA from the nucleus within one hour of HSV infection. Preliminary results indicate that this dramatic effect on the cell nucleus requires the cellular ATM DDR component, suggest- ing that this is a host response to the foreign viral DNA; and 3) epigenetic regulation of HSV gene therapy vectors. As with lytic and latent infection HSV gene therapy vectors are also regulated by host cell epigenetic mechanisms during transduction. Initial HSV gene vectors showed short-term expression due to cytotoxicity by HSV ICP0, but recent gene therapy vectors that have all viral immediate-early genes and internal inverted repeat sequences deleted and the transgene bounded by insulator sequences show long term expression of the transgene and low cytotoxicity. We have found that the viral genomic internal repeats increase replication of HSV gene therapy vectors in complementing cells and, surprisingly, increase expression of a transgene in normal cells. We will investigate these three exciting new aspects of host responses to DNA virus infection through the following specific aims: 1. Determine the mechanism(s) of viral restriction by IFN-induced levels of IFI16 in normal human fibroblasts; 2. Determine the mechanism and effects of nuclear blebbing during HSV infection; and 3. Determine the role of epigenetics and genome structure in replication of and transduction by HSV gene therapy vectors.
