Effects of GAPDH packaging defect inside virions on HIV-1 replication. (A) Effects of GAPDH siRNA treatment on CEM/LAV-1 cells. Viable cells (left) and dead cells (right) were assessed by trypan blue staining. (B) GAPDH siRNA knockdown efficiency is confirmed by western immunoblotting. Expression of GAPDH was analyzed in cell lysates from CEM/LAV-1 cells transfected with GAPDH or control siRNA 72 h after transfection. (C) Effects of GAPDH siRNA on incorporation of GAPDH and HIV-1 proteins inside virions. The incorporation of GAPDH, gp120, RT, Pr55gag, or CA was analyzed by western immunoblotting of lysates from viruses produced from CEM/LAV-1 cells transfected with GAPDH or control siRNA. The anti-GAPDH antibody and HIV-1-positive plasma were used for western immunoblotting. (D) Effects of GAPDH siRNA on virus release in CEM/LAV-1 cells transfected with GAPDH or control siRNA. The virus release in culture supernatant was directly determined by p24 ELISA. The mean values of at least three independent experiments are shown. (E) Effects of GAPDH siRNA on the incorporation of HIV genomic RNA into viral particles. The level of genomic RNA in the control virus (normalized to RT activities) was set as 100%. (F) Infectivity of GAPDH-packaging-defective virus. Infectivity was evaluated on the basis of the luciferase activity in lysates of TZM-bl cells. The value in the control experiment was set as 100%. The mean values of at least three independent experiments are shown. (G) Effect of defect in GAPDH packaging on reverse transcription in TZM-bl cells. The early strong-stop DNA products were determined by quantitative real-time PCR analysis as described in “Methods”. The significance of difference (Student’s t-test) is indicated as follows: *, p<0.01; n.s., not significant. The mean values of at least three independent experiments are shown. The error bars denote the standard deviation.