The depolymerase activity of Kip3 suppresses spindle microtubule polymerization to limit spindle midzone length and prevent microtubule buckling in response to sliding forces.
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.
Human herpesviruses (HHVs) establish lifelong latent infections, which undergo periodic reactivation and remain a major cause of morbidity and mortality, especially in immunocompromised individuals. Currently, HHV infections are treated primarily with agents that target viral DNA polymerase, including nucleoside analogs; however, long-term treatment can be complicated by the development of drug resistance.
Although it has been known for decades that HSV replication results in the formation head‐to‐tail concatermers, the mechanism by which concatemers form is not understood. One long‐standing model involves circularization of incoming viral genomes followed by rolling circle replication. However, several lines of evidence suggest that HSV DNA replication may be more complex, relying instead on a recombination‐dependent replication mechanism. The HSV single‐strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a single strand annealing protein (SSAP). HSV encodes a conserved alkaline nuclease (UL12) that interacts with ICP8 to form a complex that is reminiscent of evolutionarily conserved phage recombinases, such as lambda red a/ß. We hypothesize that ICP8 and UL12 interact to promote recombination‐dependent DNA replication to generate head‐to‐tail concatemers necessary for the production of infectious virus. Due to the multifunctional nature of ICP8, it has been difficult to tea1se out the role of ICP8 as a SSAP from the various roles it plays during viral replication as a classical SSB. We have recently identified a novel ICP8 mutant, Q706A/F707A, that lacks annealing activity but retains many of the other functions characteristic of classical SSBs. Interestingly, this mutant is unable to synthesize viral DNA, indicating that the annealing function of ICP8 is essential for viral DNA synthesis (Weerasooriya, DiScipio et al. PNAS 2018) Annealing is a complex process requiring many types of molecular interactions. We hypothesize that it involves both ICP8‐DNA and ICP8‐ICP8 interactions as well as conformational changes induced by the recognition of complementary DNA. Based on their proximity to Q706A/F707A, we suggest that annealing may be mediated by a cluster of conserved residues (residues 701 to 719) in the shoulder domain of ICP8. We have identified five conserved arginine residues (R701, R704, R712, R713 and R714) in this region. Alanine mutagenesis of these residues showed that all mutants, except R704A are unable to complement the growth of a null mutant suggesting that these residues are important for viral growth. Biochemical characterization of purified mutant and WT proteins will be presented along with models for how ICP8 promotes efficient annealing during DNA synthesis. Support or Funding Information NIH funding AI069136 and AI021747 to S.K.W and F30AI143125 to K.A.D
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