5240 Nucleic Acids Research, 2011, Vol. 39, No. 12 Figure 5. Luciferase expression is similarly affected by the uAUG in- sertions in the presence or absence of HIV-1 particle production. C33A cells were transfected with wt or mutant luciferase construct in the absence or presence of 100 ng of the pLAI molecular clone. pRL-CMV was co-transfected as an internal control. The ﬁreﬂy and Renilla luciferase activities in the lysates were determined 2 days post-transfection. Fireﬂy luciferase activity was corrected for Renilla luciferase activity. CA-p24 was determined in the supernatant and was consistent in all transfections (20.3 ng/ml average, 2.37 ng/ml standard deviation). The overall increase observed upon HIV-1 co- expression is similar as upon Tat co-expression (compare with Supplementary Figure S1). The relative representation of the data set is shown in Supplementary Figure S3. One of three independent experi- ments is shown, error bars indicate standard deviation. DISCUSSION In this study, we analysed the window of ribosomal scanning on the HIV-1 genomic RNA by introducing uAUGs throughout its 50-UTR. Insertion of uAUGs sig- niﬁcantly blocked translation initiation on the Gag start codon if the uORF was of sufﬁcient length, independent of its location. This inhibition was not observed when UAG sequences were inserted at the same positions as a control for the insertional mutagenesis. These results are in agreement with the observation that natural HIV-1 isolates do not harbour uAUGs in the 50-UTR, suggesting that uAUGs are not tolerated (1). Furthermore, the results support previous studies that investigated the effect of an uAUG insertion at a single location in the HIV 50-UTR, not only in reporter constructs but also in infectious mo- lecular clones (63,74–76). In all of these cases, expression of either viral or reporter genes was severely affected. Unfortunately, the uAUGs were inserted in the proposed IRES domain (41) and control insertions were not analysed in these studies. It was therefore uncertain if the AUG insertions destroyed IRES activity or ribosomal scanning. The ﬁndings from our study strongly support the latter explanation. Western blot analysis showed that insertion of uAUGs in various constructs (A3–A7) resulted in the translation of a Luc product initiated at the in frame Luc start codon downstream of the Gag start codon. This shift in start codon selection from AUGGag to AUGLuc was not observed with any of the control U constructs. This ob- servation strongly suggests that the uORF-translating ribosomes can resume scanning in these constructs, bypass the Gag start codon and reinitiate further down- stream. It has been well established that the efﬁciency of translation reinitiation depends on the size of the upstream ORF and the intercistronic distance (72,73,77). Thus, the constructs A1 and A2 with short ORFs and long intercistronic distances had to be re-evaluated, as the efﬁ- cient Gag-Luc expression could be due to lack of riboso- mal scanning in the region with the inserted uAUGs or to efﬁcient reinitiation and sufﬁcient intercistronic distance to allow a second-translation event on the AUGGag. Indeed, when we increased the length of the short ORFs in constructs A1–A5, A2–A5 and A2-LF, translation of Gag-Luc was severely reduced in a uAUG-dependent manner. This observation not only conﬁrms the correl- ation between ORF size and reinitiation efﬁciency (72,73,77), but strongly suggests that reinitiation accounts for the wt levels of Gag-Luc production from the constructs A1 and A2, and thus support ribosomal scanning in this region of the 50-UTR. The A7 construct also has a very short uORF, but it predominantly expresses Luc, which is likely caused by the short (20 nt) intercistronic distance between uORF7 and AUGGag. The scanning ribosomal subunit is unlikely to regain its full initiation capacity, e.g. by acquisition of essential initi- ation factors such as the eIF2–GTP–Met–tRNA ternary complex, before it reaches the Gag start codon. The intercistronic distance between uORF7 and AUGLuc is 101 nt and apparently compatible with reinitiation on the Luc start codon. HIV-1 infection may affect the translation initiation mechanism by disfavouring ribosomal scanning. HIV-1 Vpr has been suggested to induce cell cycle arrest which modestly affects scanning-dependent translation (58,59,78) and IRES activity in the HIV-1 50-UTR was shown to be increased in cell cycle-arrested lysates (41). Alternatively, the viral protease activity can cleave and inactivate the translation initiation factor eIF4G in vitro using the recombinant HIV-1 protease (50–52). eIF4G is essential for cap-dependent ribosomal scanning; it is cleaved during poliovirus infection upon which translation of cellular mRNAs ceases (79). However, translation of IRES-containing poliovirus RNA continues independent of intact eIF4G (47). A similar scenario has been postulated for HIV-1 infection (50–52). We thus co-expressed the HIV-1 proteins from an infectious mo- lecular clone with the LTR-luc reporter constructs to address ribosomal scanning under conditions that resemble HIV infection. Luciferase expression from the uAUG and UAG constructs was similar with or without HIV-1 proteins (Figure 5 and Supplementary Figure S1). Speciﬁcally, the inserted uAUGs inhibited downstream Gag-Luc expression in a similar fashion, indicating that the window of ribosomal scanning on the HIV-1 50-UTR was not affected by the viral proteins. In addition, co-expression of the poliovirus 2 A protease, which efﬁ- ciently cleaves and inactivates eIF4G (80), reduced luciferase expression from wt and deletion mutant con- structs (Supplementary Figure S4). Gene expression driven by an HCV IRES (81) was increased by 1.4-fold under these conditions, whereas a control con- struct in which the HCV IRES was inactivated was simi- larly affected by 2A protease as the HIV-1 LTR constructs. In our view, these data strongly suggest that