Ocular infections caused by herpesviruses are important clinical problems. Keratitis caused by herpes simplex virus (HSV) is a leading cause of blindness in the U.S. and sometimes require corneal transplantation to restore vision. Treatment of HSV corneal infections with topical nucleoside analogs has been efficacious in treating acute disease, but therapy for other forms of HSV-induced corneal disease and ocular disease caused by other human herpesviruses, including varicella-zoster virus (VZV) and cytomegalovirus (CMV), has not proven very successful. Treatment of HSV keratitis with combinations of nucleoside analogs like acycloguanosine (ACV) and human interferon-alpha(IFN) has been shown to produce better therapeutic response than either antiviral agents alone. We and others have shown that this combined effect reflects true synergism between these two agents at the cellular level. Synergistic antiviral activity between nucleoside analogs and IFN has been demonstrated against other members of the herpesvirus family as well. We have found evidence that IFN treatment alters the metabolism of nucleosides in HSV-infected human corneal stromal cells in such a manner that the competition between ACV and natural nucleosides for HSV-specific enzymes is shifted in favor of ACV. Our hypothesis is that this alteration in nucleoside metabolism is due to the selective repression of expression of HSV genes. We propose to determine the point in HSV replication inhibited by IFN treatment (i) by measurement of the activities of HSV-specific enzymes,(ii) by surveying the synthesis of HSV proteins using gel electrophoresis of radiolabeled infected-cell proteins,(iii) by quantitation of the production of viral mRNAs using slot blot hybridization with cloned HSV genomic DNA, and(iv) by mobility shift assays to detect the formation of HSV-cell protein/DNA complexes associated with immediate early gene transactivation. We propose that the altered nucleoside metabolism detected in IFN-treated, HSV-infected human corneal cells may reflect a modification of normal cellular nucleoside transporters. Using selective inhibitors of nucleoside transporters, we will determine the substrate specificity and number of nucleoside transporters on corneal cells and the effects of infection, IFN treatment, or combination of IFN and ACV on those transporter parameters. Using HSV as a model virus we will test our hypothesis and determine how combinations of IFN and ACV achieve synergistic antiherpes activity. We will then determine whether the synergistic anti-VZV activity we have demonstrated in corneal cells in culture is due to the same or different mechanism. Elucidation of the mechanism of those synergistic anti-herpes activity may allow therapy with combinations of nucleoside analog and IFN to be tailored to achieve maximum efficacy in the treatment of eye disease caused by human herpesviruses, including HSV, VZV, and CMV, that do not respond well to current monotherapy.
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