Figure 3

HIV-1 mRNAs associate with Argonaute 2. 293 cells were transfected with HIV-1 molecular clone pNL4-3, Myc-Ago2 or Myc-AgoPAZ9 as indicated. 48 hours later cells were harvested and cytoplasmic extracts were prepared. Total RNA was purified from a fraction of harvested cells while the rest was subjected to immunoprecipitation using anti-Myc antibody. After washing, a fraction was used to analyze the amount of Myc-Ago2 and Myc-Ago2PAZ9 immunoprecipitated by Western blotting (a), and the rest of the Myc-IPs was used for RNA extraction. HIV-1 mRNAs (TAR and unspliced), Hdm2 and GAPDH mRNA were quantified from total RNA (b, left panel) or from Myc immunoprecipitated mRNPs (b, right panel) by RT-PCR using specific oligonucleotides. c) Experiment was performed as in fig 3 except that 293 cells were transfected with HIV-1 ΔPSP which contains a partial gag/pol deletion but retains all the mRNA splicing sites [66], and 32P-labelled nucleotides were used in the PCR reaction. PCR products were visualized by autoradiography.