Molecular analysis of the cytoplasmic domain of CD4: overlapping but noncompetitive requirement for lck association and down-regulation by Nef.

Abstract

Expression of the HIV Nef protein results in the down-regulation of cell surface expression of CD4, with a di-leucine motif in the cytoplasmic domain of CD4 being required for this effect. However, our results indicate that this motif is not sufficient to confer sensitivity to down-regulation by Nef. Using site-directed mutagenesis and a transient expression system, we demonstrate that an alpha-helical stretch of amino acids of the cytoplasmic tail of CD4 is also required for the down-regulation of CD4 induced by Nef. Some CD4 mutations allowed the discrimination between PMA- and Nef-induced down-regulation, suggesting the existence of multiple pathways. In addition, our results demonstrate that this motif is involved in the association of CD4 with the tyrosine kinase p56lck, thus defining a multifunctional domain of CD4. Although there is overlap between the sequence requirement for lck association and susceptibility to Nef, we fail to detect any preferential decrease in lck association with CD4 when Nef is expressed during acute HIV infection. Altogether, these results demonstrate that there is an overlapping, but noncompetitive, sequence requirement in the cytoplasmic domain of CD4 for lck association and down-regulation by Nef.