FIG. 4.

Determining the sensitivity of incoming genomic HIV-1 RNA to siRNA degradation. (A) The env gene of v120-A or v126-D was inserted into the self-inactivating lentiviral vector pMND-GFPpre, which contains an internal MND promoter driving GFP, the central polypurine tract (cPPT)/central termination sequence, and the HIV-1 LTR with U3 deleted and cytomegalovirus promoter added at the 5′ LTR. PBS, primer binding sequence; wPRE, Woodchuck hepatitis virus posttranscriptional regulatory element. The virus particles derived from triple transfections of 293T cells with the packaging vector (pCMVΔR8.91), the VSV-G-pseudotyping vector (pMD.G), and pMND.GFPpre, pGFPenv120-A (containing the env target sequence of v120-D), or pGFPenv126-D (containing the env target sequence of v126-D). The viral RNA in these virus particles can be reverse transcribed, and the proviral DNA can be integrated into the target 293T cells. However, the LTR in the integrated virus genome cannot drive transcription and thus does not express viral RNA with the env target sequence. An internal promoter in the integrated virus genome can transcribe an mRNA encoding enhanced GFP. Eight graphs describe the flow cytometry results gated for 293T cells expressing GFP (M2) from the vGFP-env120-A (B) and vGFP-env126-D (C) infections in the presence or absence of siRNA120a or siRNA126a. Quantitative results from this experiment are shown in panel D. All experiments were performed in triplicate.