ATRX limits the accessibility of histone H3-occupied HSV genomes during lytic infection

Histones are rapidly loaded on the HSV genome upon entry into the nucleus of human fibroblasts, but the effects of histone loading on viral replication have not been fully defined. We showed recently that ATRX is dispensable for de novo deposition of H3 to HSV genomes after nuclear entry but restricted infection through maintenance of viral heterochromatin. To further investigate the roles that ATRX and other histone H3 chaperones play in restriction of HSV, we infected human fibroblasts that were systematically depleted of nuclear H3 chaperones. We found that the ATRX/DAXX complex is unique among nuclear H3 chaperones in its capacity to restrict ICP0-null HSV infection. Only depletion of ATRX significantly alleviated restriction of viral replication. Interestingly, no individual nuclear H3 chaperone was required for deposition of H3 onto input viral genomes, suggesting that during lytic infection, H3 deposition may occur through multiple pathways. ChIP-seq for total histone H3 in control and ATRX-KO cells infected with ICP0-null HSV showed that HSV DNA is loaded with high levels of histones across the entire viral genome. Despite high levels of H3, ATAC-seq analysis revealed that HSV DNA is highly accessible, especially in regions of high GC content, and is not organized largely into ordered nucleosomes during lytic infection. ATRX reduced accessibility of viral DNA to the activity of a TN5 transposase and enhanced accumulation of viral DNA fragment sizes associated with nucleosome-like structures. Together, these findings support a model in which ATRX restricts viral infection by altering the structure of histone H3-loaded viral chromatin that reduces viral DNA accessibility for transcription. High GC rich regions of the HSV genome, especially the S component inverted repeats of the HSV-1 genome, show increased accessibility, which may lead to increased ability to transcribe the IE genes encoded in these regions during initiation of infection. Author summary The genome of herpes simplex virus (HSV) is subject to host cell processes that organize DNA into ordered structures, known as chromatin, and function to regulate gene expression. Shortly after entry to the host cell nucleus, HSV DNA becomes associated with histones, protein subunits of nucleosomes around which DNA is wrapped. We evaluated the ability of known histone regulatory proteins to load histones onto HSV DNA. Only combinatorial depletion of these factors could reduce the levels of histones on viral DNA, revealing that multiple histone chaperone pathways promote chromatin loading on HSV DNA. However, one of the histone-loaders, ATRX, was significantly more potent in restricting HSV replication than the other factors. While ATRX is widely known for its role in maintaining cellular DNA stability, it is also known to act as a general restriction factor for DNA viruses. To investigate the unique mechanism by which ATRX restricts HSV, we used a high-resolution sequencing-based assay to assess the accessibility of HSV DNA. We found that ATRX promotes reduced accessibility of viral DNA during infection, likely preventing access for factors necessary for viral gene expression and replication. Our study provides new insight into the mechanism by which ATRX acts a generalized DNA virus restriction factor.