Development of successful drugs, therapeutics, or vaccines for AIDS requires insight into the pathogenic mechanisms of HIV. The molecular events that mediate HIV-induced cytopathology have not yet been elucidated. However, new studies indicate that prior investigations underestimated the in vivo load of HIV virion components and cells chronically-infected with HIV by several orders of magnitude. Therefore, direct killing by HIV could contribute to depletion of CD4+ T-cells in persons with AIDS. The investigators observed that HIV mediates dramatic changes in the activities of plasma membrane-associated ion transport systems. One manifestation of these changes is ballooning degeneration of single and syncytial cells. The first specific aim is to identify which HIV proteins are involved in alteration of plasma membrane-associated transport systems using site-directed mutants and HIV variants that differ in the ability to induce cytopathology. The env gene products, SU (gpl20) and TM (gp4l) have been implicated in HIV cytopathogenesis. The availability of a number of mutants in HIV env proteins provide powerful tools for dissecting the effects of various domains within these proteins on ion transport processes. One possibility suggested by their prior studies is that certain HIV proteins may themselves form or interact with existing plasma membrane channel proteins. Therefore, as a part of this aim they will microinject mRNA encoding the wild-type or mutant HIV env proteins into Xenopus oocytes and other eukaryotic expression systems to define which individual HIV proteins or protein domains influence ion fluxes. The second specific aim is to determine the molecular mechanisms by which HIV induces an increase in the activity of the Na+/K+/2Cl- contransport system. Preliminary results demonstrate that blocking the activity of this volume regulatory ion transport system prevents development of HIV-mediated cytopathology. Direct measurements of the levels of the cotransporter and its mRNAs are proposed using newly available monoclonal antibodies and cDNAs. Another focus of the proposed research are cells that survive the acute cytopathic effects of HIV to form persistently-infected cultures.
The third aim i s to define physiological differences involving plasma membrane transport systems between cells that undergo acute cytopathology and cells that survive HIV infection to form persistently-infected cells. The investigators will define the molecular mechanisms by which cells persistently-infected with HIV are resistant to ouabain, a specific inhibitor of the Na+/K+ATPase (sodium pump). Direct measurements of the levels of the sodium pump and its mRNAs are proposed. The investigator believes that the proposed studies will provide new information regarding mechanisms of the direct cytotoxic effects of HIV virion components.
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