Herpes simplex virus (HSV) infection often results in a recurrent disease that manifests as skin lesions in the facial or genital area. Herpes virus infections are a leading cause of blindness and fatal endemic enecphalitis. Reactivation of herpes is a serious problem for individuals with immune systems compromised by chemotherapy or HIV infection. Productive infection and the consequent cell destruction by herpes virus requires activation of viral gene expression by a viral protein designated ICP4 (IE175, Vmw175). Initial characterization of the structure and function of ICP4 suggests that it is an ideal target for anti-viral drugs. ICP4 has been conceptually divided into 5 regions on the basis of comparative sequence analysis with related members of the alphaherpesvirus family. These proteins share extensive homology in regions 2 and 4. There is evidence for a transactivation domain in region 1, a DNA binding domain in region 2, and nuclear localization signal in region 3. Although all 5 domains are required for full ICP4 activity, no specific functions have been assigned to regions 4 and 5. The long-term goal of this investigation is to define the structural basis for the functions of ICP4.
The specific aims of this project are (1) to characterize the ICP4 during productive infection; and (4) to investigate interactions between ICP4 and a component of the 20S proteasome. The roles of TAD-1 in productive infection and pathogenesis will be determined by infection of cells and animals with a TAD-1 negative mutant. TAD-1 will be further defined by analysis of the effect of point mutations in TAD-1 on transactivation. Coimmunoprecipitation assays with mutant proteins will be performed to define the residues in region 4 that are responsible for TBP/TFIIB interactions. The biological significance of the TBP/TFIIB binding sites in region 4 will be investigated by complementation assays and analysis of viral mutants. Interactions between ICP4 and proteasomes will be investigated in transfected and infected cells. The results of these analysis will be integrated into a working model for ICP4.
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