Abstact

Herpes simplex virus type 1 (HSV-1) causes lifelong infections in human cells and is associated with a range of diseases. HSV-1 DNA replication requires seven viral proteins, including the major DNA-binding protein ICP8, the origin-binding protein UL9, and proteins that comprise the helicase-primase and DNA polymerase complexes. UL9 functions as a DNA helicase that specifically recognizes and binds to the viral origins of replication, OriS and OriL. Here we report the cryo-EM structure of the UL9/ICP8/DNA/ATPgammaS complex at an overall resolution of 3.18 A. This structure revealed that UL9 employs an alpha-helix to separate the DNA strands and captures the initial step of OriS unwinding, in which the C-terminal domain of UL9 specifically binds to the major groove of a DNA double helix, while the N-terminal helicase domain engages the unwound leading and lagging strands. ICP8 interacts with the extreme C-terminal region of UL9, preventing UL9 dimerization. Simultaneously, it binds and stabilizes the leading-strand DNA adjacent to UL9. Together, these findings provide mechanistic insight into UL9-driven DNA unwinding and the cooperative action of UL9 and ICP8 at HSV-1 replication origins, establishing a structural framework for the rational interpretation of prior biochemical data and for the design of new antiviral drugs.