Nucleic Acids Research, 2011, Vol. 39, No. 12 5237 Figure 3. Insertion of uAUGs in the HIV-1 50-UTR differentially affects protein expression levels. (A) Overview of uAUGs and upstream open reading frames in A1–A8. The A1–A8 reporter constructs encode uORFs that differ in size (indicated in nt). Triangles indicate the positions of the uAUGs. The 30 border of the uORFs is determined by endogenous stop codons in the HIV-1 50-UTR. The intercistronic distances vary among the constructs and are also indicated (in nt). The A8 uORF and Gag-Luc open reading frame are overlapping. (B and C) C33A cells were transfected with wt or mutant luciferase construct and pcDNA-tat. pRL-CMV was co-transfected as an internal control. The ﬁreﬂy and Renilla luciferase activities in cell lysates were determined two day post transfection. Fireﬂy luciferase activity was corrected for Renilla luciferase activity. Wt expression levels are set as 1. One of the four independent experiments is shown, error bars indicate standard deviation. (D and E) C33A cells were transfected with wt or mutant luciferase reporter, pcDNA-tat and pRL-CMV. Fireﬂy and Renilla luciferase activities were determined and equal amounts of Renilla luciferase activity were applied to SDS–PAGE. Luciferase protein was detected using western blot analysis with anti-luciferase antibodies. The constructs express two luciferase products: the Gag-luciferase fusion protein (Gag-Luc) and the non-fused luciferase (Luc) protein. M is mock-transfected cells. mRNA levels did not deviate signiﬁcantly from the wt mRNA level after quantiﬁcation of several independent assays (Figure 2B). Therefore, the luciferase mRNA levels were not signiﬁcantly affected by the introduction of the uAUGs, in agreement with studies on other mRNAs (70,71). We proceeded to analyse luciferase expression from the A1–A8 mutant luciferase constructs. The constructs were transfected into C33A cells and luciferase activity was measured after 2 days. A construct expressing Renilla luciferase was co-transfected as an internal control. The results show that luciferase expressed from the A5 and A8 constructs decreased 5-fold compared to wt (Figure 3B). A reproducible 2-fold inhibition of luciferase activity was observed for the A3, A4 and A6 constructs. The A1 and A2 constructs expressed similar luciferase activity as wt, whereas the A7 expression level was reproducibly increased. Luciferase was expressed in the presence of the viral Tat protein to activate LTR-driven tran- scription. Experiments carried out in the absence of Tat co-expression showed a general 4-fold reduction in luciferase activity, but we observed the same relative ex- pression pattern for the wt and mutants (Supplementary Figure S1). As the mutational effects could be a consequence of the sequence insertion and not per se of the introduction of the start codon, we also inserted control sequences (A CCUAGG) at all positions. These control inserts are of the same nucleotide composition as the uAUG inserts, but the AUG start codon is inactivated into UAG. Expression of these U constructs was similar to wt (Figure 3C and Supplementary Figure S2), which indicates that the change in luciferase expression from A3 to A8 as compared to U3–U8 is caused by the inserted uAUG