HSV-1, one of nine human herpesviruses (HHVs), has infected more than 3.7 billion people under the age of 50 (67% of the world?s population) and is associated with recurrent cold sores, genital lesions, keratitis, corneal blindness, encephalitis and disseminated neonatal infections. The ability of herpesviruses to establish latent infections that undergo periodic reactivation significantly contributes to their ability to cause life-long disease, viral shedding and transmission to new hosts, underscoring the critical need for a better understanding of their mechanism of DNA replication. Despite the identification of seven viral proteins necessary for HSV DNA synthesis and three origins of replication, the current understanding of the mechanism of viral DNA replication is woefully incomplete. Although it has been known for decades that HSV replicates by concatemer formation, the mechanism by which concatemers form has yet to be identified. Several lines of evidence suggest that HSV promotes a unique form of DNA replication that utilizes a recombination-dependent mechanism to produce concatemers. We have identified a two component HSV recombinase comprised of a 5? to 3? exonuclease (UL12) and a ssDNA annealing protein or SSAP (ICP8) (exo/SSAP). This complex is reminiscent of the ? phage Red ?/? recombinase required for recombination dependent replication in ? phage and in vivo recombination-mediated genetic engineering (recombineering). Interestingly exo/SSAP recombinases are conserved in most dsDNA viruses of bacteria, protozoa, plants and mammals that replicate by concatemer formation. HSV DNA replication is further complicated by the fact that the viral genome contains nicks and gaps, and the introduction this damaged genome is known to activate cellular DNA damage response (DDR) pathways, some of which are antiviral. Thus, HSV has two major challenges: to produce concatemers that can be packaged while at the same time avoiding cellular antiviral processes. ICP8 is a central player in all stages of the DNA replication process, acting as a hub for protein-protein interactions necessary to initiate viral DNA synthesis, bind cooperatively to ssDNA and promote single strand annealing.
In Aim 1 we will explore ICP8 interactions required for initiation of DNA replication and cooperative binding to ssDNA.
In Aim 2 we will explore the role of ICP8 and UL12 in annealing and recombination-dependent DNA replication. We have shown that the annealing activity of ICP8 is essential for viral DNA replication during infection, supporting the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.
In Aim 3 we will examine the role of UL12 in the manipulation of host DDR pathways. Herpesviruses have evolved an unusual strategy to replicate their genomes that is distinct from bacterial and cellular DNA replication mechanisms. However, the remarkable degree of conservation between viruses that replicate through concatemers underscores the fact that these viruses have evolved an extremely successful strategy utilizing recombination-mediated mechanisms for replication of difficult to replicate genomes.

Public Health Relevance

Herpes Simplex Virus Types 1 and 2 (HSV-1 and HSV-2), are ubiquitous human pathogens responsible for oral, genital and sight-threatening ocular infections that can be life threatening even in immunocompetent adults. In individuals with compromised immune systems, HSV infections are often fatal. Our goal is to contribute to the understanding of mechanisms underlying replication of this virus and to provide information that will lead to novel and effective antiviral therapies.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Beisel, Christopher E
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University of Connecticut
Schools of Medicine
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Grady, Lorry M; Szczepaniak, Renata; Murelli, Ryan P et al. (2017) The exonuclease activity of HSV-1 UL12 is required for the production of viral DNA that can be packaged to produce infectious virus. J Virol :
Bermek, Oya; Weller, Sandra K; Griffith, Jack D (2017) The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks. J Biol Chem 292:15611-15621
Darwish, Anthar S; Grady, Lorry M; Bai, Ping et al. (2016) ICP8 Filament Formation Is Essential for Replication Compartment Formation during Herpes Simplex Virus Infection. J Virol 90:2561-70
Lou, Dianne I; Kim, Eui Tae; Meyerson, Nicholas R et al. (2016) An Intrinsically Disordered Region of the DNA Repair Protein Nbs1 Is a Species-Specific Barrier to Herpes Simplex Virus 1 in Primates. Cell Host Microbe 20:178-88
Smith, Samantha; Weller, Sandra K (2015) HSV-I and the cellular DNA damage response. Future Virol 10:383-397
Mossman, Karen L; Weller, Sandra K (2015) HSV cheats the executioner. Cell Host Microbe 17:148-51
Pozhidaeva, Alexandra K; Mohni, Kareem N; Dhe-Paganon, Sirano et al. (2015) Structural Characterization of Interaction between Human Ubiquitin-specific Protease 7 and Immediate-Early Protein ICP0 of Herpes Simplex Virus-1. J Biol Chem 290:22907-18
Smith, Samantha; Reuven, Nina; Mohni, Kareem N et al. (2014) Structure of the herpes simplex virus 1 genome: manipulation of nicks and gaps can abrogate infectivity and alter the cellular DNA damage response. J Virol 88:10146-56
Weller, Sandra K; Sawitzke, James A (2014) Recombination promoted by DNA viruses: phage ? to herpes simplex virus. Annu Rev Microbiol 68:237-58
Grady, Lorry M; Bai, Ping; Weller, Sandra K (2014) HSV-1 protein expression using recombinant baculoviruses. Methods Mol Biol 1144:293-304

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