5236 Nucleic Acids Research, 2011, Vol. 39, No. 12 sulphate (SDS)–polyacrylamide gel electophoresis (PAGE) to analyse the ﬁreﬂy luciferase proteins produced. Proteins were subsequently blotted on a low-autoﬂuorescence membrane, incubated with a poly- clonal antibody against luciferase (1:1000) (Promega). Bound antibodies were visualized with horse radish peroxidase-linked (HRP) donkey anti-goat IgG and the ECL+ kit (Amsersham Biosciences). The resulting lumi- nescent signal was analysed after exposure of the blot to X-ray ﬁlm for between 10 s to 5 min. RESULTS Design of the uAUG blocks How translation initiation proceeds on HIV-1 genomic RNA has thus far not been answered conclusively. Controversy remains over whether the HIV-1 genomic RNA contains an IRES element (41,42) or not (62,63). This prompted us to address this issue from a different angle. We created a ﬁreﬂy luciferase reporter construct with the HIV-1 genomic 50-UTR including the Gag start codon. Luciferase gene expression is driven from the HIV-1 LTR promoter, which can be enhanced by the addition of the viral Tat protein. The constructs contain the 50 75 nt of the Gag ORF fused to the luciferase ORF, which allows the formation of long distance interactions between sequences in the 50-UTR and Gag ORF (24). As a consequence, the reporter constructs contain two in frame start codons: AUGGag and AUGLuc (Figure 1B). This allowed the detection of ribosomes that scan past the Gag start codon. We subsequently introduced uAUG blocks at eight positions in the 50-UTR (A1–A8; indicated with red ﬂags in Figure 1A). In the case of scanning, these uAUGs will reduce the expression of the downstream reporter gene, since the authentic start codon will not be reached by the scanning ribosome. However, if the uAUGs are inserted upstream of an active IRES element they will not affect gene expression. The inserted uAUGs were ﬂanked with the consensus Kozak sequence (ACCA UGG) to allow optimal recognition by scanning riboso- mal subunits. Wild-type (wt) HIV-1 nucleotides were used where possible to create the Kozak consensus, thus minimizing the number of inserted nucleotides (marked in red in Figure 1A). A single nucleotide was mutated in the A2 construct to comply with the 3 purine require- ment (indicated with a black box in Figure 1A). We introduced the uAUG blocks in regions of the 50-UTR predicted to be single stranded in order to minimize dis- ruption of the RNA structure, except for A2, in which the U5-AUG duplex is likely affected. However, ablation of this structure does not affect translation (25). Introduction of uAUGs affects Gag translation in a site-speciﬁc manner To rule out that the uAUG insertions affected LTR- driven transcription and/or RNA stability in our test system, we ﬁrst determined the luciferase mRNA levels in C33A cells transfected with the wt and mutant reporter constructs (Figure 2). Total RNA was isolated 2 days after transfection and the luciferase mRNA was reverse transcribed with a 32P end-labelled oligonucleotide primer that anneals to the gag coding sequence. The radio-labelled cDNA products were run on a denaturing sequence gel (Figure 2A). A modest decrease in mRNA levels was observed for the mutants A1–A6, but the Figure 2. Luciferase mRNA levels are not signiﬁcantly affected by the uAUG insertions. Wild-type (wt) and mutant constructs were transfected into C33A cells, total RNA was isolated and subjected to primer extension reactions. cDNA products were analysed on a denaturing polyacrylamide gel (panel A) and quantiﬁed (panel B). The wt mRNA level was set at 1. A representative of two independent experiments is shown. The error bars indicate standard deviation.