We next assessed the effect of TAR on the frameshift efficiency. TAR (Figure 2A ) was inserted at about 40 nt from the 5 0 end of the mRNA in pDual-HIV, generating pDual-HIV-TAR(-1) and (0) ( Figure 2B ). We avoided placing TAR at the very end of the mRNA, since such a position could interfere with the binding of the 40S subunit to the messenger (23,26,27 and references therein). We first examined the effect of a high amount of TAR that inhibits PKR and stimulates translation initiation (21) . The frameshift efficiency was assessed in Jurkat and 293T cells. When 2 mg of pDual-HIV-TAR were delivered into the cells, the frameshift efficiency was decreased to 70% of its value in absence of TAR in either Jurkat or 293T cells ( Figure 2C and D) . Under the conditions of these assays, the frameshift efficiency in absence of TAR was 6.1 AE 0.2% in Jurkat cells and 11.3 AE 0.9% in 293T cells. These values, and the value of 5.1 AE 0.4% observed in the experiment described in the preceding section with Jurkat cells that were transfected under slightly different conditions (see details in 'Materials and Methods' section), are comparable to the values obtained with different heterologous systems containing the HIV-1 frameshift region, which were shown to range between 2 and 10% in mammalian cultured cells (8, 22, 28, 29) . It can be recalled here that several groups observed that the absolute value of the frameshift efficiencies changes, depending upon various parameters such as the conditions used for the assay and the type of cultured cells (30) . We then investigated whether the decrease in frameshift efficiency observed with pDual-HIV-TAR was influenced by the position of TAR in cis or in trans from the reporter mRNA. Two other constructs were used, pDual-HIV-50TAR, where the distance between TAR and the 5 0 end of the reporter mRNA was increased by 50 nt compared to pDual-HIV-TAR, and pTAR, that provides TAR in trans from the reporter mRNA expressed from pDual-HIV ( Figure 2B ). The frameshift efficiency was decreased to 75 and 60%, respectively, in Jurkat cells and 293T cells transfected with pDual-HIV-50TAR compared to the value in absence of TAR. When Jurkat and 293T cells were co-transfected with 2 mg of pDual-HIV and 2 mg of pTAR, the frameshift efficiency was reduced to 70% of its value in absence of TAR, a decrease similar to that observed when TAR was present in cis of the reporter mRNA ( Figure 2C and D) . These results indicate that it is the presence of TAR in the cells and not its presence in the reporter mRNA that decreases HIV-1 frameshift efficiency. The effect of TAR on the frameshift efficiency was confirmed when using an infection system to deliver the reporters into the cells (see Figure 1 in the Supplementary Data).