Project Summary Following primary ocular HSV-1 infection, the virus replicates in the eye and establishes latency in the trigeminal ganglia (TG). In a latently infected individual, the virus can occasionally reactivate and travel back to the eye causing recurrent disease. Reactivation from latency is the major cause of eye disease. However, the mechanisms underlying this process are not well defined, and currently it is not clear whether reactivation reflects a failure in latency mechanisms or independent mechanisms that break latency. It is well established that the continued expression of the HSV-1 Latency Associated Transcript (LAT) is a characteristic of latency, and this is associated with suppression of apoptosis and regulation of T cell responses to the infected sensory neurons. Recently, we found that in the TG of mice infected with LAT(-) virus, the levels of the HSV-1 receptor, HVEM, but not other HSV-1 receptors was significantly down-regulated. Furthermore, HSV-1 latency and reactivation were reduced significantly in HVEM-/- mice as compared to wild-type mice. Notably, the LAT function in upregulating HVEM mapped to two recently described small non-coding LAT RNAs (sncRNA1 and 2), since HVEM was upregulated following transient transfection with plasmids expressing either small non- coding RNA. In parallel, we have found that viral glycoprotein gD, capable of binding HVEM and triggering its signaling through NF-κB, is expressed at low levels in latently infected TG. Collectively, our preliminary data provide compelling evidence supporting the hypothesis that LAT can enhance latency-reactivation and T-cell exhaustion by upregulating HVEM, which in turn promotes HSV-1 reactivation, possibly by facilitating the binding of gD to HVEM. Although the LAT(-) virus expressing baculovirus inhibitor of apoptosis protein gene (cpIAP) had similar levels of latency as wild-type LAT(+) virus, this recombinant virus did not increase HVEM levels suggesting that this reactivation mechanism does not promote reactivation by regulating responsiveness to apoptosis. Rather, this mechanism appears to interfere with the ability of LAT to promote immune evasion. We propose to test this hypothesis using in vivo analyses of engineered recombinant viruses to: (1) Test whether binding of HSV-1 gD to HVEM is required for efficient reactivation in TG of latently infected mice; and (2) Test whether the LAT sncRNAs upregulate HVEM by binding to the HVEM promoter. Validation of this hypothetical model will identify previously undescribed mechanisms that contribute to HSV-1 reactivation and will provide the framework for identification of molecular targets and viral immune evasion response that could be exploited to better manage latent HSV infection. CLINICAL SIGNIFICANCE AND