The long-term goal of this translational research project is to improve survival of HIV/HCV co-infected patients by finding new pharmaceutical targets in HCV and identifying new viral products that can be used in vaccines and in diagnostic and prognostic tests. HCV-associated liver disease is a leading cause of death among HIV/HCV co-infected patients. Using custom bioinformatics tools, we found several RNA structural elements, and of utmost significance, discovered an alternate reading frame, whose products, ARFPs, stimulate immune responses in both mono-infected and co-infected patients (Walewski et al., RNA, 2001 and 2002). A. Experiments in Specific Aim I test the hypothesis that ARF expression and the immune response to ARFPs are modulated during the course of disease progression and by treatment with interferon. ELISAs will be used to detect and titer anti-ARFP antibodies in co-infected and mono-infected patients. Proliferation assays will be used to measure T cell responses. Western blots will be used to determine the sizes of core/ARFP proteins in both non-tumor and HCC liver specimens; EIA and QRT-PCR will be used to quantify core antigen and HCV RNA levels, respectively. Immunohistochemistry will be used to localize core protein and ARFPs in liver specimens. B. Experiments in Specific Aim II test the hypothesis that ARF expression is regulated by RNA structures that stimulate recoding events. ARF is in the +1 reading frame. Expression of other +1 reading frame genes that encode auto-regulatory factors, such as antizyme, is stimulated by specific RNA structural elements, which are similar to those in the core/ARF gene region. We will use in vitro translation and directed mutagenesis to determine the role of RNA elements in ARF expression. C. Experiments in Specific Aim III test the hypothesis that core and ARFPs have distinct biological properties, and that ARFPs stimulate cell growth. Gene expression profiling, cell cycle measurements, and oxidative stress responses will be used to differentiate the effects of core and ARFPs in transfected Tet-Off HepG2 cells. Western blots, immunoprecipitation, and mass spectroscopy will be used to determine the sizes of core/ARF gene products. ARFP-specific poly- and monoclonal antibodies will be developed to determine ARFPs' subcellular location. The yeast two-hybrid system will identify human proteins that bind ARFPs. These studies will identify new targets for therapeutics and vaccine development.
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