ANALYSIS OF HIV-1 CELL ENTRY USING CHEMICALLY INDUCED T CELL MUTANTS

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We have previously described the isolation of EMS-induced CEM mutants which express intermediate levels of surface CD4 (50-75% of wild type expression), yet differ markedly from wild type CEM cells as well as from other mutants with similar CD4 expression in their susceptibility to HIV 1 infection. These mutants produced reduced number of syncytia following infection with the gpl6O-vaccinia vector (VSC25). Furthermore,,infectivity assays using HIV 1 (MN strain), showed a significant reduction in their infectibility, as judged by virus titrations, longterm RT assays, and quantitative PCR (ar 72 hr post infection). Two of these mutants were found to respond abnormally to induction with the PKC activator TPA. Similar to wild type CEM, they down regulated their surface CD4 by 50% within 1 hr of TPA treatment. In contrast, TPA treatment for 24 hr. induces expression of CD4 and CD25 (IL2R) on the parental line as well as other mutants, but not on these two mutants. TPA induced phosphorylation of membrane receptors was found to be normal for CD4 and CD5, but not for CD7. CAT assays using the HIV 1 LTR (139)-CAT plasmid + TPA induction were completely negative for these two mutants. Transfection with control plasmids, e.g., Actin-CAT and RSV-CAT resulted in CAT activity. Interestingly, cotransfection with LTR-CAT + pCV1 (tat) did not result in transactivation of the CAT gene via the TAR element. Gel retardation assay using the NF-k binding oligo and nuclear extracts from TPA and TNF-induced cells, showed induction of NF-k binding protein in the mutants, but at a significantly ,reduced levels. Further biochemical analyses of these mutants would allow identification of cellular gene(s) which are important for optimal HIV I infection land are dependent on the PKC phosphorylation pathway.