Interferons (IFN) are proteins produced by cells in response to virus infection. There are three major IFN types, alpha, beta, and gamma. IFNs are not directly antiviral, but induce their antiviral actions by binding to cell receptors and inducing the production of a group of 20 or more proteins, some of which have been shown to be responsible for antiviral activity. IFNs play important roles in the response to herpes simplex virus (HSV) infections, including ocular infections causing herpetic keratitis and acute retinal necrosis. IFN-alpha combined with acyclovoir, an inhibitor of HSV DNA synthesis, produced synergistic anti-HSV activity in cornea stromal cells. In these cells IFN-alpha was found to reduce the level of several members of the early temporal class of proteins. These affected proteins are enzymes responsible for nucleoside metabolism and for viral DNA replication. Although early proteins levels are decreased, the mRNA levels which encode these enzymes were increased. Three mechanisms for this regulation will be investigated; (i) a decrease in mRNA association with polysomes, (ii) a decrease rate of translation, or (iii) an increase in the degradation of protein. The function of four IFN- inducible proteins with known antiviral action against other viruses will be studied: (i) The activity of the double-stranded RNA-activated protein kinase that phosphorylates a factor required for initiation of translation will be measured; (ii) The binding of IFN-induced RNA-binding proteins, for example 9-27, will be detected by gel shift assay; (iii) The binding of HSV early protein to the p15 protein, a protein with sequence homology to ubiquitin, the protein that binds to and targets proteins for degradation will be examined by immunoprecipitation and immunoblot; and (iv) The Mx protein that appears to function in protein transport within the cells will be assessed by immunofluorescence for co-localization with HSV proteins. The three major types of IFNs will be tested to determine whether their combined function in cornea stromal cells is synergistic with each other and with acyclovir in the inhibition of HSV replication and whether they also inhibit early protein synthesis or function by different mechanisms. Finally, we will examine the effects of IFN's on HSV replication in retinal pigment epithelial cells, a target cell for the virus infection in acute retinal necrosis. The mechanism of action of IFNs in these retinal cells will be assessed to determine whether IFNs effects are the same as in cornea stromal cells. These studies will allow us to understand how endogenously produced IFNs function to restrict HSV replication during ocular infection and how they may be used exogenously alone or with conventional antiviral agents to treat HSV ocular infections.
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