To measure HIV-1 frameshift efficiency, we used the dualluciferase reporters pDual-HIV(-1) and (0) (8) . These plasmids are derived from pcDNA3.1Hygro+ (Invitrogen) and contain the HIV-1 frameshift region inserted between the coding sequences of the Renilla (15) . The figure is adapted from Gebauer and Hentze (14) . Only the factors we refer to in the text are named. The 40S ribosomal subunit associates with the ternary complex [initiation factor 2 (eIF2) plus GTP plus the initiator tRNA, Met-tRNA i Met ] and with other factors, and binds to the 5 0 cap structure of the mRNA. This binding requires the eIF4F complex formed by three initiation factors: eIF4E, the cap-binding protein, eIF4G, a scaffold protein and eIF4A, a RNA helicase that unfolds secondary structures. After each round of initiation, eIF2 is released from the ribosome in association with GDP. Phosphorylation of the a subunit of eIF2 (eIF2-a) prevents the recycling of eIF2-GDP in eIF2-GTP, blocking translation initiation. Thapsigargin induces endoplasmic reticulum stress, which stimulates the PERK kinase that phosphorylates eIF2-a, reducing the level of functional eIF2 (55) (56) (57) . Rapamycin shuts down the mammalian target of rapamycin (mTOR) pathway, which blocks the phosphorylation of the translation repressor 4E-BP, and hypophosphorylated 4E-BP sequesters the initiation factor eIF4E (58, 59) . Hippuristanol is a selective inhibitor of eIF4A (60) , which interferes with the binding of the 40S subunit to the mRNA. luciferase (Rluc) and the firefly luciferase (Fluc). Expression of these genes is under control of a CMV promoter, which is followed by a T7 promoter. Plasmid pDual-HIV(0) differs from pDual-HIV(-1) by the addition of an adenine after the slippery sequence in the frameshift region. Derivatives of pDual-HIV(-1) and (0) were constructed where the TAR sequence was inserted after the CMV and T7 promoters. A TAR-containing fragment flanked with HindIII sites obtained from pcDNA3-RSV-TAR-Rluc plasmid (23), a kind gift from L. DesGroseillers (Universite´de Montre´al), was cloned in the HindIII site of pDual-HIV to produce pDual-HIV-TAR(-1) and (0), where the TAR sequence is located at a distance of about 40 nt from the 5 0 end of the reporter mRNA. To produce pDual-HIV-50TAR(-1) and (0), where the TAR sequence is at a larger distance from the 5 0 end of the reporter mRNA, a cassette of a 50-nt noncoding sequence was inserted in the AflII site of pDual-HIV, followed by the insertion of TAR immediately after these 50 nt, in the HindIII site. The oligonucleotides for the cassette were cass50nt-fwd and cass50nt-rev (see the sequence of all the oligonucleotides used in this study in Table 1 of the Supplementary Data). Plasmid pTAR, which expresses the free TAR sequence in trans from the reporter mRNA, was made by inserting the TARcontaining fragment flanked with HindIII sites into the HindIII restriction site of pcDNA3.1Hygro+. Derivatives of pTAR, pTARuucg Ã and pTARibulge Ã , which express mutants of TAR, were constructed by cloning oligonucleotide cassettes (cass_TAR-uucg Ã fwd and cass_TAR-uucg Ã rev or cass_TAR-bulge Ã fwd and cass_TAR-bulge Ã rev) between the two NheI restriction sites present in the TAR sequence of pTAR. In the first mutant, the upper loop, CUGGGA, is replaced with UUCG and, in the second mutant, the bulge UCU preceding the upper loop is deleted. Plasmid pCGNiC [a generous gift from N. Hernandez, Cold Spring Harbor Laboratory (24) ] expresses a mutant of the TAR-binding protein Tat (Tat Ã ), named TatC30,31A.