5232–5244 Nucleic Acids Research, 2011, Vol. 39, No. 12 doi:10.1093/nar/gkr113 Published online 9 March 2011 Ribosomal scanning on the 50-untranslated region of the human immunodeficiency virus RNA genome Ben Berkhout, Kelly Arts and Truus E. M. Abbink* Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Received October 15, 2010; Revised February 11, 2011; Accepted February 14, 2011 ABSTRACT Translation initiation on most eukaryotic mRNAs occurs via a cap-dependent scanning mechanism and its efficiency is modulated by their 50-untranslated regions (50-UTR). The human im- munodeficiency virus type 1 (HIV-1) 50-UTR contains a stable TAR hairpin directly at its 50-end, which possibly masks the cap structure. In addition, the 50-UTR is relatively long and contains several stable RNA structures that are essential for viral replication. These characteristics may interfere with ribosomal scanning and suggest that transla- tion is initiated via internal entry of ribosomes. Literature on the HIV-1 50-UTR-driven translation ini- tiation mechanism is controversial. Both scanning and internal initiation have been shown to occur in various experimental systems. To gain further insight in the translation initiation process, we determined which part of the 50-UTR is scanned. To do so, we introduced upstream AUGs at various positions across the 50-UTR and determined the effect on expression of a downstream reporter gene that was placed under control of the gag start codon. This strategy allowed us to determine the window of ribosomal scanning on the HIV-1 50-UTR. INTRODUCTION Human immunodeﬁciency virus type 1 (HIV-1) particles contain two identical full-length positive-strand RNA molecules as genome. The full-length RNA not only serves as the viral genome, but also functions as an mRNA to encode the Gag and Gag-Pol polyproteins. The highly structured 50-UTR is the most conserved part of the HIV-1 genome and is involved in several steps of the viral replication cycle (1). Distinct functions have been assigned to individual sequence and/or structure motifs (presented in different colours in Figure 1A). The 50- UTR starts with the stable TAR and the polyadenylation (polyA) hairpins. The well-characterized TAR hairpin mediates transcription activation by binding the viral Tat protein and the cellular protein cyclin T (2–10). The polyA hairpin inhibits premature polyadenylation of the nascent RNA by masking the AAUAAA polyadenylation signal (11,12). The U5 region is located downstream of the polyA signal and contains important signals for reverse transcription, such as the primer binding site (PBS) and the primer activation signal (PAS) (13). Additional motifs are located further downstream in the 50-UTR. These include the RNA dimer initiation signal (DIS), the major splice donor site (SD) required for the generation of subgenomic mRNAs and the RNA packaging signal ( ) (14–22). Previous work identiﬁed an evolutionary conserved long-distance interaction between U5 sequences and the Gag initiation codon known as the U5-AUG duplex (23,24). This duplex does not modulate the level of HIV-1 mRNA translation (25), but affects RNA dimer- ization and packaging into virus particles (26,27). The high structural complexity in the 50-UTR is expected to interfere with the efﬁciency of one very im- portant step of the viral life cycle: protein synthesis. Translation initiation on eukaryotic mRNAs is generally initiated by ribosomal scanning [reviewed in ref. (28)]. In this process, translation initiation factors interact with the 40S ribosomal subunit and the mRNA 50-cap structure. Subsequently, the ribosomal subunit migrates along the 50-UTR, until it encounters a favourable AUG start codon that marks the start of an open reading frame (ORF). Several mRNA features determine translation ef- ﬁciency: the presence and accessibility of a 50-cap structure (29–31), the length and secondary structure of the 50-UTR (31,32) and the nucleotide context of the initiation codon (33,34). The optimal context of the initiation codon in *To whom correspondence should be addressed. Tel: +44 1223 336862; Fax: +44 1223 336846; Email: truusabbink@yahoo.com Present address: Truus E. M. Abbink, Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Level 5, Hills Road, Cambridge CB2 0QQ, UK. The Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.