Our long term goal is the development of efficient clinical interventions to decrease the incidence of HSV-1 induced corneal disease and blindness. To achieve this we plan to map, characterize, and elucidate the function of the HSV-1 gene (designated the corneal tropism, or CT, gene) that allows most strains isolated from individuals with recurrent corneal disease to infect intact, unabraded cornea. In contrast, most HSV-1 strains isolated from non-ocular sites require corneal scarification for ocular infection of experimental animals. HSV-1 strains that can infect unscarified cornea tend to produce more severe experimental corneal disease than strains requiring scarification, further suggesting the CT gene's importance in corneal disease.
Our specific aims therefore include: 1. Determining and mapping the CT gene. Our preliminary rescue experiments between strains that can (McKrae) and cannot (KOS) infect unscarified cornea have already mapped this gene to within a single McKrae restriction fragment. We will continue these """"""""rescue"""""""" experiments using homologous recombination with progressively smaller defined Mckrae restriction fragments. The CT gene will be further fine mapped by deletion and/or insertion mutational analysis in McKrae. 2. Comparative sequencing and fine mapping of the corneal tropism gene. Sequencing of the gene from strains that cannot, and strains that can infect unscarified cornea will be done to look for potentially important differences. Site specific mutants will be made in regions of interest to convert that region of each strain to the sequence of the other and determine if the phenotype changes correspondingly. Conversion of KOS to the McKrae phenotype and McKrae to the KOS phenotype would conclusively prove the involvement of the gene and of the specific site. 3. Analysis of the corneal tropism gene product and its mechanism of action. To determine the corneal tropism protein's function and hence the mechanism of infection of unscarified cornea (and the mechanism by which cornea scarification allows certain strains to infect the cornea), comparative analyses will be done between CT proteins from viruses that can and cannot infect unscarified cornea, and between KOS and KOS in which the CT gene has been altered to produce virus that no longer requires scarification. The mechanism by which some HSV-1 strains can infect unscarified cornea is likely to involve more rapid viral attachment to corneal cells. If so, this study will also be important in understanding the molecular biology of viral attachment and entry. Once defined and characterized the CT gene and its protein should be excellent targets for clinical interventions, such as antivirals and/or vaccines, to decrease the incidence of HSV-1 cornea disease and blindness.
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