Beschreibung:
<jats:p>Vpu as a human‐immunodeficiency‐virus‐type‐1‐encoded 81‐amino‐acid integral‐membrane protein was expressed in <jats:italic>Escherichia coli</jats:italic> using the inducible p<jats:italic>trc</jats:italic> promoter of an ATG fusion vector. Recombinant Vpu is associated with membranes of <jats:italic>E. coli</jats:italic> and could be partially solubilized by detergents. Recombinant Vpu was phosphorylated <jats:italic>in vitro</jats:italic> with purified porcine casein kinase II (CKII) as well as with a CKII‐related protein kinase found in cytoplasmic extracts of human and hamster cells. Recombinant Vpu associated with <jats:italic>E. coli</jats:italic> membranes has turned out to be the best substrate for <jats:italic>in vitro</jats:italic> phosphorylation with CKII. This reaction can be inhibited by heparin and the ATP analogue 5,6‐dichloro‐1‐(β‐D‐ribofuranosyl)benzimidazole (DRB), both known to be potent inhibitors of CKII. Radiolabelled γATP and γGTP were used as phosphate donors for <jats:italic>in vitro</jats:italic> phosphorylation of recombinant Vpu. <jats:italic>In vivo</jats:italic> phosphorylation of Vpu‐1‐infected H9 cells was also inhibited by DRB. We concluded therefrom that the Vpu protein is phosphorylated by the ubiquitous CKII in HIV‐1‐infected human host cells.</jats:p><jats:p>Two seryl residues in the sequence of Vpu (position 52 and 56) correspond to the consensus <jats:sup>s</jats:sup>/<jats:sub>T</jats:sub>XX<jats:sup>D</jats:sup>/<jats:sub>E</jats:sub> for CKII. These potential phosphorylation sites are located within a well‐conserved dodecapeptide of Vpu (residues 47–58), which is found in different HIV‐1 strains as well as in a Vpu‐like protein of SIV<jats:sub>CPZ.</jats:sub></jats:p><jats:p>Monoclonal and polyclonal antibodies directed against two different epitopes of Vpu were used for immunoprecipitation of Vpu from HIV‐1‐infected cells and for detection of Vpu in Western blot analyses. Vpu from HIV‐1‐infected cells as well as recombinant Vpu expressed in <jats:italic>E. coli</jats:italic> were determined by SDS/PAGE using 6 M urea to be 9 kDa, which corresponds to the calculated molecular mass of Vpu.</jats:p>