Structural basis of HSV-1 DNA replication origin-binding protein UL9

The herpesvirus DNA replication machinery comprises a battery of viral enzymes that orchestrate viral genome synthesis. In HSV-1, the machinery consists of seven essential components, including the replication origin-binding protein UL9, ssDNA-binding protein ICP8, the heterodimeric DNA polymerase complex UL30/UL42, and the heterotrimeric helicase-primase complex UL5/UL8/UL52. UL9, an SF2 helicase family member, functions as a dimer that sequence-specifically recognizes the replication origin sites and unwinds duplex DNA to initiate replication. Furthermore, UL9 interacts with several viral replication components to recruit the replication machinery, as well as with cellular proteins that regulate its own functions. However, the molecule mechanisms underlying UL9s multifunctionality remain poorly understood due to the lack of structural information. In this study, we present the first cryo-EM structures of UL9 in both apo and DNA-bound states. Combining biochemical and enzymatic assays, we elucidate the molecular basis of UL9 dimerization, origin recognition and activity regulation by ICP8-binding.