Packaging of LysRS and tRNALys3is regulated by GAPDH. (A) Effect of GAPDH on enzymatic activity of HIV-1 RT. RT activity assay was performed as described in “Methods”. The value in the control experiment was set as 100%. The activity in the presence of GAPDH (RT:GAPDH ratio= 1:10 or 1:100) is shown as the activity relative to that of the control. The mean values of at least three independent experiments are shown. Packaging of (B) LysRS and (C) tRNALys3 in GAPDH-packaging-defective virus. (B) LysRS and GAPDH were detected by western immunoblotting using anti-LysRS and anti-GAPDH antibodies in lysates from viral particles produced from CEM/LAV-1 cells transfected with GAPDH or control siRNA. (C) Incorporated tRNALys3 level was determined by reverse transcription quantitative PCR analysis as described in “Methods” and normalized by viral genomic RNA level. The amount of tRNALys3 in the control virus was set as 100%. The mean values of at least three independent experiments are shown. (D) Interaction of GAPDH with Pr55gag and p160gag-pol. GAPDH was immunoprecipitated from the clarified lysate from CEM/LAV-1 cells with the anti-GAPDH antibody. The precipitated proteins were analyzed by western immunoblotting using the indicated antibodies to the proteins shown (anti-p24 antibody for Pr55gag and anti-RT antibody for p160gag-pol), and were visualized by enhanced chemiluminescence analysis. The significance of difference (Student’s t-test) is indicated as follows: *, p<0.01; n.s., not significant. The error bars denote the standard deviation. (E) The packaging of GAPDH in the enhanced-LysRS-packaging virus. The packaging efficiencies of LysRS and GAPDH were analyzed in lysates from viral particles produced from HEK293 cells cotransfected with pNL-CH and either the LysRS expression or control vector.