Mohni, Kareem N.

ABSTRACT Herpes simplex virus type 1 replicates in the nucleus of the host cell and utilizes many cellular proteins to facilitate DNA replication. HSV-1 DNA replication is closely linked with homologous recombination, and HSV-1 encodes a two-subunit recombinase consisting of an exonuclease (UL12) and a single-strand DNA-binding protein (ICP8). We identified the cellular deubiquitinating enzyme USP15 as a new UL12-binding partner. USP15 is one of the most enriched proteins on viral DNA and has recently been implicated in regulating cellular homologous recombination. Utilizing isolation of proteins on nascent DNA (iPOND) to compare the proteins on wild-type and UL12-deficient viruses, we observed that USP15 is not recruited to newly replicated DNA in the absence of UL12. We also observed that UL12 and USP15 interact directly in vitro using purified proteins. Together, these data suggest that UL12 directly recruits USP15 to viral DNA. UL12 stimulates a type of homology-directed repair called single-strand annealing (SSA). We generated a USP15 knockout cell line with an integrated GFP-based SSA reporter and observed that UL12 and USP15 both contribute to the maximal induction of SSA. In addition to the reporter cell line, there is a 10-fold reduction in the frequency of marker rescue, and individual replication forks move slower in the absence of USP15. These data support a role for USP15 in facilitating recombination during the virus life cycle and the maintenance of replication fork stability. IMPORTANCE HSV-1 is a ubiquitous pathogen in the global population that causes lifelong latent infection with sporadic reactivation in the host. It has long been appreciated that HSV-1 DNA replication exhibits high rates of recombination and is linked to the host DNA damage response. In this study, we show a novel interaction between UL12, a member of the HSV recombinase, and the deubiquitinating host enzyme USP15. We show that USP15 physically interacts with UL12 and is required for UL12 to maximally stimulate recombination. In the absence of USP15, we also observe an overall reduction in virus growth. This work demonstrated that host proteins facilitate viral-induced recombination. The interaction of USP15 with UL12 represents a potential target for intervention against HSV-1 infection and associated disease.

ABSTRACT Herpes simplex virus is a large double-strand DNA virus that replicates in the nucleus of the host cell and interacts with host DNA replication and repair proteins. The viral 5′ to 3′ alkaline nuclease, UL12, is required for production of DNA that can be packaged into infectious virus. The UL12-deleted virus, AN-1, exhibits near wild-type levels of viral DNA replication, but the DNA cannot be packaged into capsids, suggesting it is structurally aberrant. To better understand the DNA replication defect observed in AN-1, we utilized isolation of proteins on nascent DNA (iPOND), a powerful tool to study all the proteins at a DNA replication fork. Combining iPOND with stable isotope labeling of amino acids in cell culture (SILAC) allows for a quantitative assessment of protein abundance when comparing wild type to mutant replication forks. We performed five replicates of iPOND-SILAC comparing AN-1 to the wild-type virus, KOS. We observed 60 proteins that were significantly lost from AN-1 forks out of over 1,000 quantified proteins. These proteins largely represent host DNA replication proteins including MCM2-7, RFC1-5, MSH2/6, MRN, and proliferating cell nuclear antigen. These observations are reminiscent of how these proteins behave at stalled human replication forks. We also observed similar protein changes when we stalled KOS forks with hydroxyurea. Additionally, we observed a decrease in the rate of AN-1 replication fork progression at the single-molecule level. These data indicate that UL12 is required for DNA replication fork progression and that forks stall in the absence of UL12. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a near-ubiquitous pathogen within the global population, causing a lifelong latent infection with sporadic reactivation throughout the life of the host. Within at-risk and immunocompromised communities, HSV-1 infection can cause serious morbidities including herpes keratitis and encephalitis. With the possibility of herpesviruses to evade established antiviral therapies and there being no approved HSV-1 vaccine, there comes a need to investigate potential targets for intervention against infection and subsequent disease. UL12 is the viral 5'–3' exonuclease, which is required for the production of infectious progeny. In this study, we show that in the absence of UL12, viral replication fork progression is abrogated. These data point to UL12 as an attractive target for intervention, which could lead to better clinical outcomes of HSV-1-associated disease in the future.