FUNCTIONAL-ANALYSIS OF THE PHOSPHORYLATION SITES ON THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 VPU PROTEIN

The human immunodeficiency virus type 1 (HIV-1)-encoded nu pu product is a small class 1 integral membrane protein that is phosphorylated by the ubiquitous casein kinase II (CKII) in HIV-1-infected cells. The V pu protein facilitates the release of budding virions from the surface of infected cells and delays the rate of syncytium formation. In this study, we investigated the role of phosphorylation in the biological activity of Vpu. Our results show that phosphorylation of Vpu occurs o n serine residues at positions 52 and 56 located in st highly conserve d dodecapeptide sequence. Mutation of either Ser 56, or both Ser 52 an d Ser 56 impaired the ability of Vpu to delay the rate of syncytium fo rmation while retaining virion release activity at levels comparable t o nu pu(+) proviruses. Flow cytometry analysis indicates that the rela tive amounts of envelope glycoprotein gp120 expressed at the surface o f cells transfected with these nu pu mutant proviruses was two- to thr eefold greater than that observed on cells transfected with a nu pu(+) provirus. This increased expression of gp120 at the cell surface may explain the more rapid onset of syncytium formation observed in cell t ransfected with nu pu mutant proviruses. These results suggest that Vp u-facilitated virion release and delayed cytopathic effect are the con sequence of two distinct functional activities of the protein.