Determining the role of PARP14 in restricting HSV-1 replication - Project Summary Herpes simplex virus 1 (HSV-1) infects 70-90% of the human population and causes significant diseases such as blindness and life-threatening encephalitis, most notably in immunocompromised individuals. In contrast, most infections of immunocompetent hosts are asymptomatic and result in viral latency. Limited disease in most human hosts is likely the result of millions of years of co-evolution between HSV-1 and humans, due, in part, to a wide variety of host restriction factors that limit early stages of HSV-1 replication. Understanding the mechanisms used by humans to repress HSV-1 replication will help identify treatments that can mitigate severe HSV-1 diseases, especially in immunocompromised hosts. In a screen designed to identify novel host proteins that interact with the HSV-1 genome, 3 poly(ADP-ribose) polymerase (PARP) proteins were identified that bound to HSV-1 DNA with high confidence: PARP1, PARP9, and PARP14. Interestingly, prior studies had also found that these three PARP proteins were bound to HSV-1 genomic DNA, providing strong evidence that these PARP proteins could impact the replication of HSV-1, even though there have been very few reports of PARPs associated with HSV-1 replication. In addition, PARP14 has been evolving under positive selection, suggesting that it is involved in host-virus conflict, and we recently demonstrated that it can restrict coronavirus replication. Thus, the ability of HSV-1 to replicate in PARP14 knockout (KO) A549 cells was tested, and surprisingly PARP14 KO cells produced 1-2 logs more in viral yield assays and increased the plaquing efficiency ~2-logs higher compared to WT A549 cells. These results support the central hypothesis that PARP14 binds to the HSV-1 genome early in infection, leading to significant repression of HSV-1 replication. The overall objective of this application is to establish PARP14 as a novel host restriction factor and determine how it binds to the viral genome and inhibits virus replication. These objectives will be achieved through the following specific aims: 1) Establish PARP14 as a potent restriction factor for HSV-1 in cell culture and in mice; and 2) Determine how PARP14 represses the HSV-1 replication cycle and define how it binds to the viral genome. In Aim 1 a set of PARP14 knockdown/overexpressing cells and knockout mice will be used to determine if PARP14 restricts HSV- 1 replication in multiple cell culture models and in mice. In Aim 2 the stage of the viral lifecycle that is repressed by PARP14 will be determined using classical virological techniques, then ChIP-seq and several PARP14 truncation mutants will be utilized to define how it interacts with the viral genome. This research is innovative because PARP14 has not previously been described as an HSV-1 restriction factor. The proposed research is significant because it will provide a potential mechanism by which PARP14 restricts HSV-1 replication. Results from this study will lead to a greater understanding of how PARPs interact with DNA-containing viruses, which can ultimately lead to the rational development of novel therapies to prevent or treat HSV-1 diseases.
