Herpes simplex viruses are endemic in the population and are responsible for a variety of clinical diseases some of which are life threatening especially in immunocompromised individuals or in newborns. The herpes simplex virus type-1 (HSV-1) genome encodes seven viral genes that are necessary and sufficient for origin-dependent plasmid amplification in a transient transfection assay: UL30 and UL42 which specify a two subunit DNA polymerase; UL29, the single strand DNA binding protein; UL5, UL8 and UL52 which together make up a three protein helicase-primase complex and UL9 which specifies the HSV origin binding protein. UL5, 8, 9, and 52 which are the least abundant and least well studied of the replication proteins have been chosen for this study. The objective of this proposal is to carry out a detailed structure-function analysis of these four proteins in order to map domains responsible for catalytic activities, DNA binding (specific and nonspecific), sites for inter- and intramolecular interactions, nucleotide binding, and signals for intracellular localization.
The first aim i s to construct the necessary reagents to carry out all subsequent aims; these include constructs for expression of each protein in a variety of expression systems and the characterization of specific antisera.
Aim 2 is to isolate a large number of point, insertion and deletion mutations in each gene. The third and fourth aims are to carry out in vivo and in vitro characterization of mutants isolated in aim 2 for overall ability to carry out DNA replication and for specific activities such as specific and non specific DNA binding, NTP binding, DNA-dependent ATPase, helicase and primase. The nuclear localization signals on each protein will be mapped by observing localization not only of wild-type and mutant forms of each protein but also of gene fusions to cytoplasmically located proteins. The fifth aim is to locate regions on UL5, UL8 and UL52 and UL9 responsible for protein-protein interactions using genetic and biochemical approaches. Potential interactions between these proteins and other members of the replication complex will also be pursued. Genetic methods include: interference by transdominant mutations, use of a novel two-hybrid system to identify interacting proteins, isolation of second site suppressor mutations and colocalization by immunofluorescence. Biochemical methods for detecting interactions include: affinity-based methods, coimmunoprecipitation and physical methods such as glycerol gradients and gel filtration. It is anticipated that a detailed structure-function analysis of these four replication proteins will not only facilitate our understanding of the mechanisms of their action within the replication complex but may also lead to the development of novel strategies for antiviral therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021747-11
Application #
2061605
Study Section
Experimental Virology Study Section (EVR)
Project Start
1984-12-01
Project End
1997-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
11
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
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
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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