HSV INDUCED RNA DEGRADATION AND PATHOGENESIS

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Herpes simplex virus (HSV) can cause a variety of ocular diseases in humans ranging from self-limiting dendritic epithelial keratitis, conjunctivitis, and blepharitis to necrotizing stromal keratitis. HSV keratitis is a leading cause of non-traumatic blindness in the US, with approximately 500,000 cases diagnosed each year. In addition, HSV commonly causes cold sores, genital sores, and is a leading cause of viral encephalitis. The lifecyles of HSV and other neurotropic herpesviruses are characterized by a lytic phase of infection at peripheral sites during which all virus genes are expressed, and a latent phase of infection in neurons, during which gene expression is extremely limited. The switch between lytic and latent infection, however, is poorly understood. One hallmark of the neurotropic herpesviruses is their ability to shut off macromolecular synthesis in the cells they infect. It has been shown for HSV type 1 (HSV- 1) that the gene responsible for this shut off is the product of the UL41 gene known as the virion host shutoff protein or vhs. The structure and function of vhs is not understood although recent work has demonstrated homologs of vhs in HSV-2, varicella zoster virus, swine pseudorabies virus, and equine herpesvirus, suggesting that vhs may be necessary for a neurotropic lifecycle. Using HSV-1 as a model system, the objectives of this proposal are to investigate the mechanisms of action and functions of the vhs protein in the shutoff of host macromolecular synthesis in vitro and in vivo. To this end, nonsense, deletion, and substitution mutations will be introduced into the open reading frame of vhs. The effects of these mutations upon the ability of the protein to degrade host and viral mRNAs will then be measured by in vitro expression/degradation assays and in vivo following introduction of these mutations into the context of the viral genome. Resulting mutants will also be tested for their ability to establish, maintain, and reactivate from viral latency in neurons. Efforts will also be made to assess domains which are responsible for cell-specific mRNA degradation and to correlate this activity with pathogenesis. A better understanding of the functional domains of the vhs protein and its role in the regulation of HSV gene expression in acutely and latently infected cells will allow further insight into the mechanism by which HSV can persist for the lifetime of its host.