Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5′-to-3′ exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.
The repair of DNA damage is essential to maintain genomic stability. Cells have at least four distinct DNA repair pathways, and defects in any of them can lead to tumor formation and cancer progression. Herpes Simplex Virus-1 (HSV-1) manipulates components of the host DNA repair pathways. In this paper we showed that DNA repair by the single strand annealing (SSA) pathway was increased during HSV infection and that other pathways were inhibited. We also show that a viral nuclease in conjunction with either a viral or cellular single strand annealing protein can stimulate the SSA pathway. We suggest that viral DNA synthesis occurs via an SSAdependent mechanism that is reminiscent of that used by bacterial viruses such as λ. Interestingly, λ has evolved an SSA-mediated repair mechanism to exchange genetic information that has also been used to enhance gene targeting in bacteria. It is thus possible that HSV proteins could be similarly used as tools to stimulate gene targeting in human cells leading to more effective strategies for gene therapy. Furthermore, the diversity of HSV reported in human populations, combined with the high rate of genetic exchange during infection, suggests that SSA may play a role in viral evolution and pathogenesis.