A panel of mutants were derived from the human T cell line clone CEM, expressing different levels of surface CD4 receptors and demonstrating different susceptibilities to HIV-1 infection. Multiple assays were developed to analyze these clones: (a) Quantitative flow cytometry analysis of surface receptors and binding of soluble gp120. (b) Quantitative PCR to measure viral entry and proviral DNA formation. (c) Vaccinia-gp160-vector based syncytia assay. (d) Gel retardation assays to study the levels of DNA binding proteins required for optimal HIV-1 proviral reactivation. (e) Protein Kinase C enzymatic activity assay. (f) Chloramphenicol acetyl transferase (CAT) assay to measure activation of the HIV-1 LTR. Major Findings: I. No direct correlation exist between the number of surface CD4 receptors and the kinetics of HIV-1 infection. Subclones expressing less than 1000 CD4 receptors/cell were equally susceptible to HIV-1 lab strains as the parental CEM line. II. Two CEM subclones were isolated with dramatically reduced susceptibility to infection by multiple strains of HIV-1. These mutants were found to have a markedly reduced level of DNA binding protein belonging to the NFk-B family (p50). Experiments are under way to establish the underlying mechanisms for this deficiency, and to reconstitute the mutants by expression vectors containing wild type p50. The knowledge gathered in this project will be applied for the studies of neonatal T cell infection. Studies will correlate the kinetics of HIV-1 infection with induction of DNA-binding proteins, lymphokine production, and activation state.
