Detection and identification of HIV-1 RNA associated factors. A) Description of the HIV-1 constructs. Above an outline of the full-length viral genome, below the two constructs used in this work: HIVexo (carrying the MS2 binding sites after the SA7 splice site) and HIVintro (carrying the MS2 repeats in the intron). Black arrows indicate the RT-PCR primers listed in Table 2. The scheme is not drawn to scale. B) Pulldown of HIV-1 RNA and associated Tat. 293T cells expressing the indicated constructs were lysed and immunoprecipitated with anti-flag beads. Immunoblots with anti-GFP antibodies show Tat-CFP (lanes 1, 3, 5 7) and ECFPskl (lanes 5 and 7) expressed by the HIVintro construct. Tat could be immunoprecipitated only when the HIV-1 RNA is present and the association is disrupted by RNase treatment (compare lanes 6 and 8). IgH and IgL are the heavy and light chains of the immunoglobulins used in the immunoprecipitation. IP and WL stand for immunoprecipitation and whole cell's lysate, respectively. C) MS2-dependent pulldown of specific HIV-1 RNAs. U2OS clones and U2OS wt cells expressing Tat-CFP and flag-MS2nls were lysed and immunoprecipitated with anti-flag beads. RNA was extracted from immunoprecipitations and the RNA reverse-transcribed and PCR amplified with primers for β-actin mRNA (lanes 1-6), as well as with primers that differentiate spliced (lanes 7-12) and unspliced (lanes 13-18) forms of the HIV-1 RNAs which are outlined in Figure 1A.