Previous work from this laboratory has demonstarted a specific defect in cellular signaling induced in human T lymphocytes that survive after infection with the HIV-1 retrovirus. CD4 cells lose the capacity to have CD3/T cell receptor-induced intracellular calcium mobilization while calcium mobilization induced by an alternative receptor, the CD2 molecule, remains intact. The purpose of this application is to further characterize this defect. First, the defect in calcium mobilization will be examined in more detail. Antigen-specific clones and T cell hybridomas will be tested to determine if signalling is impaired after triggering by antigen as well as the CD3 complex. Digital image analysis will be used to determine in single cells if the delayed, plateau phase of calcium mobilization after CD3 stimulation is impaired by HIV-1, or if only the initial aspect of calcium signaling is affected. In addition, it will be determined if HIV-1-infection affects oscillations in calcium mobilization that occur after cellular stimulation. T cell leukemia lines will be chronically infected with the HIV-1 virus. These cell lines will be used to characterize the defect in calcium signaling by the formation of heterokaryons with the Jurkat T cell line. These heterokaryons will be analyzed to determine whether the signaling defect induced by HIV-1 is dominant or recessive. If the defect is recessive, the HIV-1 infected cells will be fused with signaling mutants of the Jurkat line and analyzed to determine if correction of the defect (complementation) occurs. Second, it will be determined if the defect in signaling occurs at the level of calcium mobilization, or if the defect is more proximal. Analysis of inositol polyphosphates will tell whether HIV-1 infected cells have impaired activation of phospholipase C. The CD3 complex will be examined for structural alterations that could account for decreased signaling ability. In specific, the relative abundance of CD3 heterodimeric zeta-eta chain and homodimeric zeta-zeta chain complexes will be determined, and the zeta chain will be studied for phosphorylation at tyrosy residues. Finally,the potential role of CD4 in the signaling defect will be examined. The signaling capacity of cells treated with monoclonal antibodies to CD4 will be compared to cells infected with HIV-1 to determine if the removal of CD4 from the surface of the cell is sufficient to induce the signaling defect. Cells will be infected with a mutant virus that causes productive infection of cells but does not cause modulation of the CD4 molecule. Thus, it will be dctermincd if the defect can be induced by components of the virus distinct from those that cause loss of the CD4 receptor from the cell surface. These approaches will clarify the mechanisms that lead to the qualitative impairments of immune function that occur after infection with HIV-1, and are therefore, important to the pathogenesis of the acquired immunodeficiency syndrome.
NIH ExPorter
NIH ExPorter