Role of herpes simplex virus polymerase DNA repair activity in viral replication

Project: Research project

Description

? DESCRIPTION (provided by applicant): All DNA based microbes including herpes simplex virus-1 (HSV-1), exhibit mutation frequencies in the order of ~0.003 mutations/replication cycle. Several processes contribute towards maintaining this level of fidelity including the nucleotide selectivity of the replicative DNA polymerase, proofreading exonuclease activity, post- replication repair to correct mis-incorporation events, and other DNA repair processes such as base excision repair. Overall, we are interested in the mechanisms that underlie genome replication and maintenance in HSV-1, their roles in the biology of the virus and their potential as targets for anti-viral therapy. HSV-1 is a large double-strand DNA virus with a genome of ~152 kbp, encoding ~75 known genes. HSV-1 is extremely widespread in the population, with up to 90% of the US population testing seropositive for the virus. Typically, the virus undergoes a productive lytic replication cycle in epithelial cells, characterized by viral gene expression, vral DNA replication and new virus production. HSV-1 persists in its host by establishing latency in sensory neurons during which there is no viral DNA replication, very limited gene expression and no virus production. Active HSV-1 infections are commonly characterized by oral lesions. In addition, herpes keratitis is a leading cause of blindness and viral encephalitis, while uncommon, is invariably fatal. The prevalence of HSV-1 and its ability to cause recurrent infections with varying degrees of morbidity and mortality make it a significant public health problem. We previously identified a novel 5' dRP and apurinic/apyrimidinic lyase activity associated with the HSV-1 DNA polymerase (UL30 protein), reported its interaction with the viral uracil DNA glycosylase (UL2 protein) and reconstituted uracil-initiated base excision repair utilizing these factors in vitro. We hypothesize that the HSV-1 base excision repair pathway involving both UL30 and UL2 is critical to viral genome maintenance, performing a critical anti-mutator role. Specifically, in this proposal we seek to address the role of the lyase activity in viral replication and its impact on replication and mutation frequency. This will be accomplished as follows: 1) Identification and mutagenesis of the UL30 lyase active site. 2) Biochemical characterization of the lyase-deficient UL30 to ensure that it is otherwise functional. 3) Construction of a lyase-deficient viral mutant. 4) Determination of replication efficiency and mutation frequency of lyase-deficient HSV-1 in cell culture. This approach will allow us to directly address our hypothesis that the UL30 lyase activity fulfills an anti-mutator function, wit the prediction that lyase-deficient HSV-1 exhibits a mutator phenotype. Demonstrating that the lyase activity of UL30 performs an anti-mutator role will provide significant new insight into the importance of DNA repair processes in HSV-1 genome maintenance. Targeting the DNA lyase activity of UL30 to cause hypermutagenesis and thereby result in the production of unfit virus during lytic replication and/or upon reactivation of latent virus may be exploited for potential drg development.
StatusFinished
Effective start/end date7/1/156/30/17

Funding

  • National Institutes of Health: $76,500.00
  • National Institutes of Health: $76,750.00

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Human Herpesvirus 1
Simplexvirus
Lyases
DNA Repair
Viruses
Mutation Rate
Maintenance
Viral DNA
Genome
DNA Replication
Uracil-DNA Glycosidase
Viral Encephalitis
Gene Expression
Exonucleases
Wit and Humor
DNA Viruses
Uracil
Viral Genes
Keratitis
Viral Genome

ASJC

  • Medicine(all)
  • Immunology and Microbiology(all)