Nucleic Acids Research, 2011, Vol. 39, No. 12 5233 Figure 1. Insertion of uAUGs in the HIV-1 50-UTR. (A) The full-length HIV-1 50-UTR encompasses nt +1 to +335 of the HIV-1 genomic RNA and harbours several structure and sequence motifs that are essential for viral replication. The RNA structure model was published previously (24). The regulatory motifs are marked in colours and further explained in the text. An asterisk indicates the Gag start codon. Red ﬂags indicate where uAUGs or UAGs are inserted. The insertions are indicated A1–A8 for the uAUG insertions. The uAUG insertions are shown in detail next to the RNA structure model; the ﬂanking Kozak sequences are underlined. The nucleotide position of the 50-UTR is indicated and red nucleotides indicate the insertions whereas the boxed nucleotide indicates a mutation. (B) The luciferase reporter construct is shown with the 50-LTR promoter elements U3, R and U5; nt positions of transcription start site (+1) and the Gag coding sequences are shown. The reporter constructs encode a fusion protein of the Gag N-terminal 25 amino acids and the ﬁreﬂy luciferase protein. As a result of the cloning strategy, the original AUG start codon that marks the beginning of the luciferase open reading frame (AUGLuc) is in frame with the Gag start codon (AUGGag). mammalian cells is the so-called Kozak sequence: RCCA UGG, in which a purine at position 3 and the G at position +4 are most inﬂuential on translation initiation efﬁciency (35). The HIV-1 50-UTR displays several characteristics that may interfere with efﬁcient scanning and hence translation initiation. Firstly, secondary structures close to the 50-end of the message can inhibit translation initiation (30,36). The 50-cap structure may be inaccessible to 40S ribosomal subunits due to the TAR and polyA hairpins at the 50-end of the RNA (Figure 1) (30,31,37). Disruption of the TAR hairpin stimulated translation efﬁciency up to 190-fold (30), indicating that the intact TAR hairpin strongly inhibits translation. Second, the 50-UTR of 335 nt is rela- tively long and highly structured (18,23,24,38–40). Obviously, viral replication occurs in spite of these