Fig. 1.

Vpu_ML supports virus particle secretion. (A) Parallel cultures of HeLa cells (2.5 × 10⁶) were transfected with 3 μg of the Vpu-defective molecular clone pNL4-3/Udel together with the Vpu-expressing constructs pNL-A1 (+Vpu), pNL-A1/Udel (−Vpu), or pNL-A1/U_ML (+Vpu_ML). Cells were labeled for 30 min with 250 μCi ³⁵S-methionine, and a pulse/chase experiment was conducted as described in the Materials and Methods section. Viral proteins from the lysates of cells and pelleted virions were immunoprecipitated with an HIV-1-reactive human serum, separated on 12% SDS-polyacrylamide gels, and analyzed by fluorography. Fluorograms depicting cell lysates (Cell) and virion fractions (SN) are shown. HIV proteins are indicated on the left. (B) p24^gag and p55^gag proteins detected in the cell lysates and the virus pellet were quantified by image analysis. The efficiency of viral particle release was calculated as the percentage of Gag proteins (p24^gag and p55^gag) present in the virus pellet relative to the total Gag proteins detected intra- and extracellularly for each timepoint and were plotted as a function of time.