Nucleic Acids Research, 2011, Vol. 39, No. 12 5239 Figure 4. The length of the uORF determines translation reinitiation efﬁciency. (A) Extension of the uORFs in A1 and A2 constructs. The A1 and A2 uAUGs were fused to the A5 uAUG, thereby creating constructs A1–A5 and A2–A5 with an uORF size of 93 nt. The U1–U5 and U2–U5 were created similarly with the U1, U2 and U5 constructs, respectively. In addition, the uORF in A2 was extended in A2-LF by insertion of a G at nt +123 (shown by an asterisk), which destroys the original stop codon. As a consequence, the uORF length is extended from 18 to 150 nt, and the intercistronic distance is decreased by 132 nt. (B and C) Transfections were performed as in Figure 3A and B. One of three independent experiments is shown. Error bars indicate standard deviation. (D and E) Western blot analysis of the luciferase proteins. See Figure 3C and D for details. Co-expression of viral proteins does not affect the HIV-1 translation initiation mechanism We thus far used a reporter construct driven by an HIV-1 promoter and the viral Tat protein was provided in trans to enhance transcription. In fact, the Tat expression vector also expressed the Rev protein, but neither protein differ- entially affected the luciferase activity expressed from the set of 50-UTR mutants. It could be argued that other viral factors modulate the translation process. For instance, it has been suggested that viral factors contribute to a cellular environment that negatively affects cap-dependent scanning, either by cleavage of eIF-4G or by induction of G2-M cycle arrest (50–53,58,59). In order to investigate whether viral trans-acting factors affect ribosomal scanning and thus the expression from the mutant reporter constructs, we repeated the transfection experi- ments in the presence of the full-length HIV-1 LAI mo- lecular clone that expresses all viral proteins. The results are shown in Figure 5 and Supplementary Figure S3. Co-expression of the molecular clone increased the luciferase expression by 3-fold, which we attribute to Tat trans-activation of the LTR promoter (compare Figure 5 with Supplementary Figure S1). The relative luciferase expression from the set of constructs did not change upon expression of HIV-1 proteins. In addition, virus production was unaltered by co-expression of wt versus mutant luciferase expression vectors.