The goal of this renewal application is to determine if blocking the mechanisms involved in intracellular HSV-1 gK processing and trafficking to the cell surface reduces virus replication and infectivity and thereby HSV-1- induced corneal scarring (CS). It is well established that HSV-1-induced CS is the result of virus-induced immune responses although the identity of the pathogenic responses is controversial. Similarly, the HSV-1 gene(s) involved in eye disease is not yet known although several lines of evidence indicate that the HSV-1 glycoprotein, gK plays a crucial role in replication and infectivity as exemplified by the inability of HSV-1 gK mutants to grow in cells lacking gK as they fail to acquire a cytoplasmic envelope efficiently and are unable to infect and establish latency in neurons. Our work shows that: (1) Deletion of gK results in reductions in virus yield, plaque size and translocation of the virus from the cytoplasm to the extracellular space; (ii) An 8mer peptide within the cleaved gK signal sequence is an immunodominant stimulatory region that upregulates T cell-responses in the cornea leading to exacerbation of CS; and (iii) Cell surface expression of gK is required for exacerbation of CS in ocularly infected mice. Thus, we examined the mechanisms that regulate gK intracellular processing and cell surface expression and in the current funding period have shown that binding of gK to signal peptide peptidase (SPP), an ER protein, is essential for HSV-1 infectivity both in vitro and in vivo. Preliminary dat further suggest that the 5' region of gK mediates its binding to SPP and binding of gK to HSV-1 UL20 mediates gK surface expression and is required for HSV-1-induced CS. These data suggest novel potential therapeutic targets for prevention of ocular HSV-1 infection and HSV-1-induced CS. We will test a hypothetical model in which the pathogenic effects of gK during primary ocular infection are mediated by two inter-related mechanisms: (i) gK binds to SPP leading to higher levels of virus replication; and (ii) gK binds to UL20, which is required for the surface expression of gK necessary for gK-induced CS and greater levels of eye disease. We will: (1) Test whether SPP plays a major role in HSV-1 infectivity in vitro and in vivo by: (i) Fin mapping the region of gK involved in SPP binding, and construction of a recombinant HSV-1 expressing a mutant form of gK that does not bind to SPP to determine if it fails to exacerbate CS in mice; and (ii) generation of SPP knockout mice to determine if they are protected from eye disease, virus replication and latency. (2) Test whether binding of UL20 to gK and the Golgi-specific DHHC zinc finger protein (GODZ), are required for proper cell surface expression of gK. We will determine if UL20 binds to gK and if UL20 binds to GODZ using pull-down assays and immunostaining; determine the function of GODZ in vitro and in vivo using GODZ knockout mice that we have generated; and determine the effects of disruption of these key binding events, alone and in combination, on the intracellular localization of gK and its cleaved products. Preliminary data is presented that indicate the feasibility of the proposed approaches. CLINICAL SIGNIFICANCE: HSV-1-induced CS can lead to blindness and is the leading cause of infectious blindness in developed countries but there is no FDA approved drug for its treatment. The proposed studies represent an entirely novel approach to the development of effective strategies for the prevention and treatment of this disease.
HSV-1 causes eye disease and can lead to blindness. Our data indicate that HSV-1 glycoprotein K (gK) binds to signal peptide peptidase (SPP) and UL20, while UL20 binds to Golgi-specific DHHC zinc finger protein (GODZ). These interactions are regulating gK expression at the cell surface of HSV infected cells. We will use several approaches to determine if blocking these interactions reduce virus replication in the eye and hence, the occurrence of eye disease. Our studies are expected to provide a novel therapeutic strategy to control vision loss from HSV-1 infection.
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