FIG. 7.

siRNA degradation on different HIV-1 mRNA species in the presence or absence of inhibitors of nuclear mRNA export. Panel A provides a schematic illustration of the location of the RT-PCR primers in the HIV-1 genome and in specific HIV-1 unspliced and multiply spliced mRNA species. Images of the agarose gels containing the various amplified HIV-1 RNA products are shown in panel B. RNA for these RT-PCRs was extracted from pNL4-3/env120-A-transfected 293T cells exposed to siRNA120a in the presence or absence of inhibitors of the CRM-1 nuclear export pathway (leptomycin B [LepB] and camptothecin [Camp]). The relative levels of these various HIV-1 RNA messages (measured by RT-PCR as described for panels A and B) in the absence of siRNAs (set to 100%) were compared to levels in the presence of siRNA and with no inhibitor of nuclear export (C), leptomycin B (D), or camptothecin (E). All experiments whose results are shown in panels A through E were performed in triplicate. Since variance was less than 10% for relative virus production and siRNA inhibition, RT-PCR amplifications were performed only on a set of serially diluted RNA samples. Panel F provides a comparison of HIV-1 RNA levels determined by using semiquantitative PCR and real-time PCR methods (SYBR green). (G) The levels of HIV-1 RNA were determined by real-time PCR for each replicate in this triplicate experiment. The levels of HIV-1 RNA were measured in the absence of nuclear export inhibitor, with leptomycin B, or with camptothecin by using the RNA protection method described in Materials and Methods. Two probes specific for HIV-1 RU5 (unspliced RNA) or U3R products were used in this RPA. semi-quant, semiquantitative.