The herpes simplex virus alkaline nuclease is required to maintain replication fork progression

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.