PUTATIVE ALPHA-HELICAL STRUCTURES IN THE HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1 VPU PROTEIN AND CD4 ARE INVOLVED IN BINDING AND DEGRADATION OFTHE CD4 MOLECULE

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a 16- kDa class I integral membrane phosphoprotein with an N-terminal membra ne-spanning region and a C-terminal cytoplasmic domain, In the cytopla smic domain, two amphipathic alpha-helices joined by a flexible turn c ontaining two phosphoacceptor sites have been predicted, Previous stud ies have shown that Vpu downregulates CD? molecules by inducing their specific degradation in the endoplasmic reticulum, Phosphorylation of serine residues 52 and 56, present within the cytoplasmic domain of th e Vpu protein, has been shown to be essential to this Vpu function, Ho wever, the contribution of these two phosphoacceptor sites in the mech anism of CD4 degradation remains undefined, Interestingly, a specific interaction between Vpu and CD4 was recently demonstrated in coimmunop recipitation experiments, Binding of Vpu was shown to be necessary but not sufficient to mediate CD4 degradation, indicating that interactio n between Vpu and CD4 represents an early step critical in triggering a process leading to CD4 degradation, To delineate the sequence(s) and /or structural determinant(s) involved in this Vpu-CD4 interaction and in the Vpu-mediated CD4 degradation, we performed a mutational analys is of the cytoplasmic domain of CD4 and Vpu, Coimmunoprecipitation exp eriments reveal that disruption of the putative alpha-helical structur e in the membrane-proximal cytoplasmic domain of CD4 affects the bindi ng to Vpu, suggesting that this structure may act as an interface for the CD4-Vpu interaction that mediates CD4 degradation, Vpu proteins co ntaining mutations in either or both of the phosphoacceptor sites (Ser 52 or/and Ser56) were inactive in regard to CD4 degradation yet retain ed the capacity to interact with the cytoplasmic domain of CD4, In an attempt to define the minimal region responsible for this interaction, we tested a panel of mutations which were designed to affect the inte grity of the putative alpha-helices present in the cytoplasmic domain of Vpu, Our results indicate that although both C-terminal alpha-helic es are required for degradation of CD4, only alpha-helix I, located in the membrane-proximal cytoplasmic region of VPu, is involved in the i nteraction between Vpu and CD4, Taken together, these results demonstr ate that alpha-helical structures in the HIV-1 Vpu and CD4 proteins ar e involved in binding and degradation of CD4 molecules.