5238 Nucleic Acids Research, 2011, Vol. 39, No. 12 start codon. These results are supportive of ribosomal scanning as a means of translation initiation on this region of the HIV-1 50-UTR. The reproducibly increased A7 expression over the U7 control remains to be explained. The reporter constructs contain two in frame AUG codons that mark the beginning of the overlapping Gag-Luc and Luc ORF (Figure 1B). We assumed that the Gag start codon was used predominantly. However, the downstream AUGLuc could be used upon leaky scanning over the Gag start codon. Both Gag-Luc and Luc will display luciferase activity, although the amino terminal extension in Gag-Luc may affect the enzymatic activity in an unknown manner. We therefore wanted to conﬁrm the luciferase activity results by western blot analysis that separates the Gag-Luc and Luc proteins. The results are shown in Figure 3D and E. Expression of the wt construct resulted in a protein cor- responding to the estimated Gag-Luc size, migrating slower in the gel than the unfused Luc product from a control plasmid in which the Gag start codon was inactivated (data not shown). The A1 and A2 constructs expressed wt levels of Gag-Luc protein, in agreement with the unaltered luciferase activity. However, introduction of the uAUGs at positions 3–8 resulted in a gradual shift from Gag-Luc to Luc protein expression (Figure 3D). The shift was caused by the inserted uAUG and not by the insertional mutagenesis itself as the U control con- structs predominantly expressed Gag-Luc (Figure 3E and Supplementary Figure S2). The inhibition of Gag-Luc expression by the inserted uAUG codons in the A3–A8 constructs was thus higher than we concluded from the results of the luciferase activity assays. It appears that the position of the introduced uAUG codon has an effect on which start codon is selected further downstream. A map of the uORFs in the uAUG constructs (Figure 3A) indicates that the Gag-Luc to Luc shift was most pronounced when the uORF terminated in close proximity of AUGGag, for instance compare A1 and A2 with A5, A6 and A7. This Gag-Luc to Luc shift could possibly be explained by translation reinitiation. This process has been studied in various systems and its efﬁ- ciency has been reported to largely depend on two vari- ables: the length of the uORF and the distance between the uORF and the downstream start codon. It has been postulated that upon translation of a short uORF, the 40S ribosomal subunit can resume scanning and reinitiate on a downstream start codon (72,73). For this second initiation event, the intercistronic distance needs to be a minimal size to allow the 40S ribosomal subunit to acquire the initiation factors required for a new round of translation (72). The intercistronic distance differs among the mutant reporter constructs, varying from 269 to 10 nt for the A1 and A5 constructs, respectively (Figure 3A). For the A1 and A2 constructs the intercistronic distance between the uORF and the AUGGag is relatively long, which can explain the predominant expression of the Gag-Luc product. In contrast, the AUGGag is bypassed in the A3–A7 constructs with shorter intercistronic distances because the ribosomal subunit may not yet be equipped for a second initiation event when it encounters AUGGag. It is not until after scanning to AUGLuc that the ribosomal subunit has acquired the translation initiation factors and can embark upon a new round of translation. This move to AUGLuc would increase the length of the intercistronic region by 81 nt. It is not surprising that the A8 construct does not express detectable levels of Gag-Luc, since its uORF overlaps with the gag coding sequences. To express the Gag-Luc protein from this construct, reinitiating ribosomal subunits must scan in the 30–50 dir- ection. The data, therefore, strongly suggest that scanning occurs on the HIV-1 50-UTR from nt 152 onwards, as construct A3–A8 all reduce translation initiation on the AUGGag. Extension of uORF1 and 2 also results in effective inhibition of Gag translation A second variable of reinitiation efﬁciency is the length of the uORF (72,73). This could explain why the level of reinitiation is not similar for all the constructs. The lengths of the uORFs in the A1–A8 constructs are shown in Figure 4. The A1, A2 and A7 constructs have very small uORFs (15–18 nt) that potentially allow efﬁ- cient reinitiation, consistent with the high luciferase ex- pression measured. The remaining constructs harbour uORFs of 39–171 nt in size that would decrease the reinitiation efﬁciency substantially. Overall, these data suggest that the insertion of short uORFs allows high ex- pression levels of the downstream reporter gene, whereas lengthening of the uORFs leads to a signiﬁcant reduction. Thus, efﬁcient Gag-Luc expression by the A1 and A2 mutants could be due to the combination of short uORFs and long intercistronic distances. To investigate these parameters, we extended the uORFs in A1 and A2, which consequently reduced the length of the intercistronic distance, by fusing these uAUGs with that of A5, thereby deleting the 50-UTR sequences in between (A1–A5 and A2–A5 in Figure 4A). A similar fusion was made between U1/U2 and U5 as control con- structs. In a separate A2 extension, we inserted a G nucleotide into the stop codon of the uORF, which causes a frameshift and extension of the uORF from 18 to 150 nt and reduced the intercistronic distance by 132 nt (A2-LF in Figure 4A). For this construct, a U control (U2-LF) was created. All constructs were analysed for luciferase expression in a luciferase activity assay and on western blot (Figure 4B–E and Supplementary Figure S2). Both analyses are in full agreement. Extension of the uORF in A1–A5, A2–A5 and A2-LF signiﬁcantly reduced luciferase activity and Gag-Luc expression, yet induced the expression of the further downstream Luc ORF. These effects are not observed with the U control constructs. These data strongly suggest that translation reinitiation is very efﬁcient when the uORFs are short as in A1 and A2, thus frustrating our strategy to determine the window of ribosomal scanning with the initial set of A1 and A2 mutants. However, extension of the uORF as in A1–A5, A2–A5 and A2-LF caused a severe Gag-Luc expression defect. These results are in agreement with a model in which the ribosome scans throughout the 50-UTR across the position of A1–A8.