ATRX Mediated Restriction of Herpes Simplex Virus 1

Abstract

A key regulator of gene expression and chromosome stability during development, replication, and genome maintenance, ATRX also restricts invading DNA viruses. We undertook a series of experiments to investigate the mechanisms by which ATRX affects viral gene regulation, formation of viral heterochromatin, and restriction of DNA virus replication using herpes simplex virus (HSV). We used herpes simplex virus to investigate the role of ATRX in gene silencing and de novo heterochromatin formation on repeat rich, nucleosome-free DNA in vivo. Viruses with bioorthogonally labeled genomes were used to detect colocalization of HSV DNA with PML, IFI16, ATRX at PML-nuclear bodies within minutes of infection. ATRX depletion by siRNA and CRISPR-Cas9 mediated knockout resulted in elevated mRNA accumulation, early viral DNA synthesis, and unexpectedly revealed that ATRX was not required for initial histone deposition or modification to HSV DNA. Rather, ATRX was required for heterochromatin maintenance on input viral DNA during times of chromatin stress. We next investigated the roles other H3 chaperones play in restriction of HSV. Using siRNAs to systematically deplete human fibroblasts of nuclear H3 chaperones, we found that the ATRX/DAXX complex is unique among nuclear H3 chaperones in its capacity to restrict HSV infection and that H3 deposition to HSV DNA occurs in a highly redundant manner. ChIP-seq for total H3 showed that the entire HSV genome was loaded with high levels of H3, but HSV in ATRX-KO cells exhibited reduced H3 accumulation. Furthermore, ATAC-seq analysis revealed that HSV DNA was not largely organized into full nucleosomes during lytic infection, and ATRX-KO globally increases viral DNA accessibility and a delayed progression of infection. Together, these findings support a model in which the unique restrictive activity of ATRX is mediated through enhanced chromatin stability, reduced viral gene accessibility, and delayed viral kinetics during lytic HSV infection.