DEGRADATION OF CD4 INDUCED BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VPUPROTEIN - A PREDICTED ALPHA-HELIX STRUCTURE IN THE PROXIMAL CYTOPLASMIC REGION OF CD4 CONTRIBUTES TO VPU SENSITIVITY

The HIV-1-encoded Vpu protein induces a rapid and specific degradation of CD4 molecules in the endoplasmic reticulum (ER). In this study, Vp u-induced degradation of CD4 in the ER was investigated by quantitativ e immunoprecipitation of CD4 following cotransfection of COS-7 cells w ith CD4 and Vpu expressors in the presence of brefeldin A, a drug that blocks protein transport from the ER to the Golgi complex. In order t o precisely define the sequence(s) or structural element(s) in the CD4 cytoplasmic domain necessary for Vpu-induced degradation, a panel of deletion and substitution mutants in the cytoplasmic domain of CD4 was generated and analyzed. In agreement with previous reports, our delet ion analysis indicates that a region encompassing amino acids 411 to 4 19 (KRLLSEKKT) in the cytoplasmic domain of CD4 was required to confer Vpu sensitivity. However, six specific substitution mutations within this region did not confer CD4 resistance to Vpu, suggesting that neit her the amino acid sequence nor the charge of the amino acids in this region was critical to Vpu-induced CD4 degradation. A dileucine motif that is important for internalization of CD4 and Nef-induced CD4 down- regulation was also not required for Vpu-induced CD4 degradation. Inte restingly, two substitution mutants (CD4EMKL and CD4MK407, 11PP) locat ed in a more proximal cytoplasmic region of CD4 abolished Vpu-induced CD4 degradation. Computer-assisted analysis of the substitution and de letion mutants conferring CD4 resistance to Vpu-induced degradation in dicated that these mutations disrupted a putative alpha-helix formed i n the proximal cytoplasmic region of CD4. Taken together, these studie s strongly suggest that a structural element in the proximal cytoplasm ic region of CD4 contributes to Vpu sensitivity. (C) 1995 Academic Pre ss, Inc.