intact eIF4G is required for optimal translation of HIV-1 RNAs and do not support the presence of an efﬁcient IRES element in the 50-UTR, in agreement with earlier data (63). IRES activities observed in various mRNAs have recently been attributed to artefacts of the experi- mental set up: cryptic promoter activity in the reporter constructs or aberrant splicing of the bicistronic RNAs resulted in the expression of unwanted subgenomic, monocistronic RNAs that were efﬁciently translated via the ribosomal scanning mechanism (78,82–88). Careful re-examining of the proposed HIV-1 IRES activity is therefore much desired (78). An alternative mechanism for translation initiation could be ribosomal shunting. Shunting is a discontinuous form of scanning in which 40S ribosomal subunits ‘jump’ from an upstream shunt donor region to a downstream shunt acceptor in the 50-UTR thus bypassing stable RNA structures and uAUGs (89). We showed that all uAUG insertions affected downstream Gag-Luc production if the uORF size was of sufﬁcient length. None of the inserted uAUGs seemed to be skipped by ribosomal subunits via a shunting mechanism. The results therefore do not provide evidence for ribosomal shunting on HIV-1 mRNAs. In conclusion, we propose ribosomal scanning as the main mechanism for the translation initiation process on HIV-1 mRNA, not only in the absence of HIV-1 proteins, but also in the presence of Tat and all other HIV-1 proteins. This leaves the question of how the relatively long and structured HIV-1 50-UTR allows ribosomal scanning and translation of the Gag and Gag-Pol structural proteins. The 50 TAR hairpin has been shown to inhibit translation in rabbit reticulocyte lysates in cis, likely by blocking cap recognition (31). It has been shown in various studies that length and structures in 50-UTRs are more inhibitory in rabbit reticulocyte lysates than in cells (90–93). In agree- ment with this, several host factors have been identiﬁed that alleviate the TAR-mediated block of translation in cells (94–96). In addition, the cellular RNA helicase DDX3 that is involved in HIV-1 RNA nuclear export (97) was recently implicated in translation regulation: its RNA helicase activity improved translation of mRNAs with long and structured 50-UTRs, possibly by unwinding secondary structure or facilitating ribosomal scanning (98). This role of DDX3 in HIV-1-driven translation has not been carefully investigated. A role in facilitating trans- lation efﬁciency on the HIV-1 50-UTR was recently sug- gested for RNA helicase A (RHA) also (99). RHA has already been described as a post-transcriptional enhan- cer for other retroviral mRNAs (100). Furthermore, DHX29 has been reported to promote translation initi- ation on mRNAs that contain highly structured 50-UTRs (101). The involvement of the various cellular factors in HIV-driven translation is an intriguing topic that warrants further investigation. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. Nucleic Acids Research, 2011, Vol. 39, No. 12 5241 ACKNOWLEDGEMENTS We owe many thanks to Wim van Est for artwork. Prosper Rek and Suuske Popma are acknowledged for technical assistance. We owe many thanks to Dr Peter Bredenbeek (Leiden University, the Netherlands) for the gift of the bicistronic HCV constructs and the 2A-expression constructs, to Dr Emily Manktelow for critically reading this manuscript and to Dr Adri A.M. Thomas for fruitful discussions. FUNDING Funding for open access charge: TOP grant from the Netherlands Organization for Scientiﬁc Research (NWO) Chemical Sciences and a VICI grant from the Netherlands Organization for Health Research and Development (ZonMW). Conﬂict of interest statement. None declared. REFERENCES 1. Berkhout,B. (1996) Structure and function of the human immunodeﬁciency virus leader RNA. Prog Nucleic Acid Res. Mol. Biol., 54, 1–34. 2. Puglisi,J.D., Tan,R., Calnan,B.J., Frankel,A.D. and Williamson,J.R. (1992) Conformation of the TAR RNA-arginine complex by NMR spectroscopy. Science, 257, 76–80. 3. Aboul-ela,F., Karn,J. and Varani,G. (1996) Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge. Nucleic Acids Res., 24, 3974–3981. 4. Aboul-ela,F., Karn,J. and Varani,G. (1995) The structure of the human immunodeﬁciency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein. J. Mol. Biol., 253, 313–332. 5. Ippolito,J.A. and Steitz,T.A. (1998) A 1.3-A resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation. Proc. Natl Acad. Sci. USA, 95, 9819–9824. 6. Berkhout,B. (1992) Structural features in TAR RNA of human and simian immunodeﬁciency viruses: a phylogenetic analysis. Nucleic Acids Res., 20, 27–31. 7. Klaver,B. and Berkhout,B. (1994) Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus. Embo J., 13, 2650–2659. 8. Dingwall,C., Ernberg,I., Gait,M.J., Green,S.M., Heaphy,S., Karn,J., Lowe,A.D., Singh,M., Skinner,M.A. and Valerio,R. (1989) Human immunodeﬁciency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro. Proc. Natl Acad. Sci. USA, 86, 6925–6929. 9. Wei,P., Garber,M.E., Fang,S.M., Fischer,W.H. and Jones,K.A. (1998) A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-afﬁnity, loop-speciﬁc binding to TAR RNA. Cell, 92, 451–462. 10. Berkhout,B., Silverman,R.H. and Jeang,K.T. (1989) Tat trans-activates the human immunodeﬁciency virus through a nascent RNA target. Cell, 59, 273–282. 11. Klasens,B.I., Das,A.T. and Berkhout,B. (1998) Inhibition of polyadenylation by stable RNA secondary structure. Nucleic Acids Res., 26, 1870–1876. 12. Klasens,B.I., Thiesen,M., Virtanen,A. and Berkhout,B. (1999) The ability of the HIV-1 AAUAAA signal to bind polyadenylation factors is controlled by local RNA structure. Nucleic Acids Res., 27, 446–454. 13. Abbink,T.E. and Berkhout,B. (2007) HIV-1 reverse transcription: close encounters between the viral genome and a cellular tRNA. Adv. Pharmacol., 55, 99–135.