Herpes simplex virus (HSV-1) is an important human pathogen responsible for self-limiting mucocutaneous lesions in immunocompetent patients and potentially lethal infections in neonates and other immunocompromised individuals. In this proposal we will test the hypothesis that HSV utilizes recombination-dependent replication to replicate its genome using both viral and cellular proteins. We propose that HSV has evolved to interact with the cellular repair and recombination machinery which the cell normally uses to respond to DNA damage and other stress factors. The cellular machinery designed to monitor and repair damaged DNA is essential for maintaining genomic stability. Mammalian cells exposed to DNA damaging agents induce cell cycle checkpoints and DNA repair pathways that serve to protect the cell from mutations and genomic rearrangements. Defects in these pathways lead to diseases such as cancer. Herpesviruses have coevolved with their hosts and developed fascinating ways of side stepping, subverting and in some cases benefiting from host cell responses. In this proposal we will test the hypothesis that HSV uses the homologous recombination (HR) repair pathway for its own benefit. Our preliminary work suggests that HSV uses a combination of viral and cellular proteins to carry out recombination-dependent replication needed to generate progeny genomes. We have previously demonstrated that the viral 5' to 3' exonuclease, UL12 and the major single strand DNA binding protein, ICP8, can function as a two-subunit recombinase. We propose to continue our studies of this viral recombinase and to test the hypothesis that HSV viral and cellular pathways for replication of its genome.
Aim 1 will test the hypothesis that recombination is essential for productive viral infection;
Aim 2 will test the hypothesis that ICP8 and UL12 work together during infection;
and Aim 3 will test the hypothesis that cellular recombination proteins are recruited to and interact with viral preprelicative sites and active replication centers. A combination of genetic, biophysical, biochemical and cell biological approaches will be used. HSV-1 is a major human pathogen, and little is known about the mechanism of DNA replication. We have proposed that the virus utilizes some of the same machinery that cells use for preventing genetic instability and cancer. Since DNA replication is a major target for antiviral drugs, it is important to fully understand the key players in this process in order to develop better strategies for treatment. In this proposal we will test the hypothesis that HSV uses a combination of viral and cellular proteins to carry out DNA replication. ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI069136-01
Application #
7079573
Study Section
Virology - A Study Section (VIRA)
Program Officer
Beisel, Christopher E
Project Start
2006-01-15
Project End
2010-12-31
Budget Start
2006-01-15
Budget End
2006-12-31
Support Year
1
Fiscal Year
2006
Total Cost
$284,000
Indirect Cost
Name
University of Connecticut
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
022254226
City
Farmington
State
CT
Country
United States
Zip Code
06030
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
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
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
Mohni, Kareem N; Dee, Alexander R; Smith, Samantha et al. (2013) Efficient herpes simplex virus 1 replication requires cellular ATR pathway proteins. J Virol 87:531-42
Mohni, Kareem N; Smith, Samantha; Dee, Alexander R et al. (2013) Herpes simplex virus type 1 single strand DNA binding protein and helicase/primase complex disable cellular ATR signaling. PLoS Pathog 9:e1003652

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