Varicella‐Zoster virus ORF9 is an antagonist of the DNA sensor cGAS

Varicella‐Zoster virus (VZV) causes chickenpox and shingles. Although the infection is associated with severe morbidity in some individuals, molecular mechanisms that determine innate immune responses remain poorly defined. We found that the cGAS/STING DNA sensing pathway was required for type I interferon (IFN) induction during VZV infection and that recognition of VZV by cGAS restricted its replication. Screening of a VZV ORF expression library identified the essential VZV tegument protein ORF9 as a cGAS antagonist. Ectopically or virally expressed ORF9 bound to endogenous cGAS leading to reduced type I IFN responses to transfected DNA. Confocal microscopy revealed co‐localisation of cGAS and ORF9. ORF9 and cGAS also interacted directly in a cell‐free system and phase‐separated together with DNA. Furthermore, ORF9 inhibited cGAMP production by cGAS. Taken together, these results reveal the importance of the cGAS/STING DNA sensing pathway for VZV recognition and identify a VZV immune antagonist that partially but directly interferes with DNA sensing via cGAS. Synopsis Molecular mechanisms that determine innate immune responses to Varicella‐Zoster Virus (VZV) infection remain poorly defined. This work reveals a key role for the cGAS/STING DNA sensing pathway, which is countered by a viral immunosuppressor. The DNA sensor cGAS and its adaptor STING detect VZV infection and induce type I interferons in infected THP1 cells. The VZV protein ORF9 blocks DNA sensing by cGAS. ORF9 interacts with cGAS and inhibits the synthesis of the second messenger cGAMP. ORF9 and cGAS phase‐separate together with DNA in vitro . Graphical Abstract The DNA sensing cGAS/STING pathway plays a key role in detecting VZV infection and is in turn directly countered by a viral immunosuppressor.