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Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Significance Herpes simplex virus (HSV) is a ubiquitous human pathogen. The mechanism of HSV DNA replication is poorly understood. ICP8, the multifunctional HSV single-strand DNA binding protein (SSB), is essential for viral growth and DNA replication. ICP8 is also capable of efficiently annealing complementary ssDNA and has been classified as a single-strand annealing protein (SSAP); however, the role of annealing during infection is still unclear. In this paper, we characterize a mutant of ICP8 that retains many of its functions as a replicative SSB but lacks annealing activity. We show that ICP8 annealing activity is essential for viral DNA replication. These findings support our hypothesis that HSV-1 uses recombination-dependent mechanisms to replicate its DNA. Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase–primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.