The role of ATRX in both promoting the establishment of HSV latency and restricting reactivation

The human pathogen, Herpes Simplex Virus (HSV), establishes latency only in neurons and can reactivate to result in the production of infectious virus for transmission to a new host. Reactivation can result in lesions at the body surface and encephalitis, in addition to potentially promoting the development of neurodegenerative

disease. Our previous work has found that the latent HSV genome is associated with repressive heterochro- matin. Therefore, entry and maintenance of HSV latency are likely regulated by the nuclear environment in the neurons, although specific proteins that function in neurons to promote heterochromatin-based silencing of the

viral genome are not known. We have found that the heterochromatin-associated protein, Alpha-Thalasse- mia/mental Retardation X-linked (ATRX), is highly abundant in neurons. ATRX is a multi-functional, hetero- chromatin-associated protein, and mutations in ATRX are associated with neurodevelopment disease. The role

of ATRX in neuronal development suggests a specific role for ATRX in modulating gene expression in neu- rons. Importantly, we have found that ATRX is required to limit HSV gene expression in neurons and promote entry into latency. Once latency is established, we have found that HSV genomes are associated with ATRX,

and these genomes are also enriched for histone H3 tri-methyl lysine 9 (H3K9me3). Furthermore, we have found that ATRX acts as a barrier to HSV reactivation in response to cell stress. The goals of this project are to determine the mechanistic functions of ATRX in regulating different stages of HSV latency. Based on our pre-

liminary data, we hypothesize that ATRX promotes the formation of H3K9 methylation to limit gene expression and promote entry into latency (tested in aim 1). We also hypothesize that once latency is established, ATRX binds to a sub-population of viral genomes that are enriched for the H3K9me3 modification and prevents lytic

gene expression occurring from these genomes (tested in aim 2). We will use our expertise in animal models of HSV latency, in addition to in vitro models of HSV latency and reactivation established in our lab, to test these hypotheses. In aim 1, we will determine the contribution of ATRX in deposition and spreading of the

H3K9me3 modification, in addition to the role of H3K9me3 in promoting entry into latency. In aim 2, we will de- termine the mechanistic function of ATRX in preventing transcription from H3K9me3-associated genomes in response to multiple reactivation stimuli. Therefore, this proposal is significant and innovative because it will

mechanistically determine the contribution of an abundant neuronal protein to both promoting HSV latency and preventing reactivation. Understanding this is crucial because it may be possible to utilize the role of ATRX to develop therapies that prevent HSV reactivation. Given that mutations to the H3K9me3 reading domain of

ATRX are associated with neurodevelopmental diseases, understanding the mechanism by which ATRX regu- lates gene expression in neurons could lead to broader treatments beyond HSV.