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.