Effect of Rac1-dependent cell signaling and the Wave2 multicomplex on virus assembly and mature VLP production. (A to D) Effect of Dia1, Pak2, Wave2, IRSp53, and Arp3 depletion on virus assembly and mature particle production. Jurkat T cells were transfected with p8.2 (Gag, Gag-Pol, and viral accessory proteins) and with the siRNA control or siRNA against Dia1, Pak2, Wave2, IRSp53, or Arp3. (A) Immunoblot analysis of HIV-1 pr55Gag and CAp24 in cell lysates and in virus particles. Tubulin was used as a loading control. (B) Extracellular virus production measured by quantification of immunoblot images, corresponding to the ratio of extracellular CAp24 to intracellular pr55Gag and CAp24. Bars show mean values and standard deviations resulting from three independent experiments. The statistical significances of differences were calculated by an unpaired t test. **, P value of <0.01; *, P value of <0.05. (C) Cell viability measured by using trypan blue. (D) Quantification of protein depletion after siRNA treatment. Bars show mean values and standard deviations resulting from three independent experiments. (E to H) Effect of the Rac1-Wave2-IRSp53-Arp3 cell signaling pathway on virus assembly and mature particle production in primary T lymphocytes (PBLs). PBLs were transfected with p8.2 and with the siRNA control or siRNA against Rac1, IRSp53, Wave2, or Arp3. (E) Immunoblot analysis of HIV-1 pr55Gag and CAp24 in cell lysates and in virus particles. Tubulin was used as a loading control. (F) Extracellular virus production measured as described above for panel C. Bars show mean values and standard deviations resulting from two independent experiments. (G) Cell viability measured by using trypan blue. (H) Quantification of protein depletion after siRNA treatment.