Vallejos M , Carvajal F , Pino K , Navarrete C , Ferres M , Huidobro-Toro JP , Sargueil B , López-Lastra M .

	Figure 1. Translational activity of the natural HIV-1 5′UTR variants isolated from clinical samples.(A) Schematic representation of the bicistronic vectors (mRNAs) used in this study. Vectors dl ΔEMCV and dl HIV-1 IRES have been previously described [10], [25]. The HIV-1 5′UTRs recovered from the HXB2 infectious clone or from RNA isolated from clinical samples were cloned into dual luciferase bicistronic (dl) vectors harboring Renilla (RLuc) and Firefly (FLuc) luciferases as reporter genes to generate the dl VAR and dl HXB2 vectors. The dl VAR vectors were arbitrarily numbered 1-13. The 5′UTR recovered from the HIV-1 infectious clone pNL4.3, dl HIV-1 IRES, was used as the reference IRES [10], while the RLuc/FLuc bicistronic vector harboring a defective encephalomyocarditis virus (ΔEMCV) IRES, dl ΔEMCV, was used as a negative control [10], [25]. (B) Bicistronic vectors were transfected into HeLa cells, which were processed 24 h thereafter. RLuc and FLuc activities were measured and the [(FLuc/RLuc)] ratio was used as an index of IRES activity, with the [(FLuc/RLuc)] ratio of the dl HIV-1 IRES arbitrarily defined as 100%. Values are the means +/- SEM from three independent experiments.
	Figure 2. The full length bicistronic RNA is expressed from the dl VAR vectors.(A) Schematic representation of the experimental procedure to detect the full length bicistronic mRNA showing the primers and the size of the expected amplicons [23]. (B and D) HeLa cells were transfected with 200 ng of the dl HIV-1, dl ΔEMCV, or the different dl VAR plasmids. Total RNA was extracted from transfected cells and quantified. (B) Extracted RNA (3 µg) was used as template in a one-step RT-PCR designed to specifically detect the bicistronic RNAs (lanes 4, 6, 8, 10, 14, and 16). To assay for DNA contamination the same reaction was conducted in the absence of reverse transcriptase (lanes 5, 7, 9, 11, 13, 15, 17). In vitro transcribed dl HIV-1 IRES RNAs (lane 2) and water (lane 3) were included as RT-PCR controls. (C) Schematic representation of the experimental procedure to detect the RLuc-RNA and FLuc-RNA showing the primers and the size of the expected amplicons (D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmol)/RNA-RLuc (pmol) ratio was calculated. Values are the means +/- SEM from three independent experiments (each RNA sample was amplified in three independent reactions).
	Figure 3. Assessment of IRES activity present within the HIV-1 VAR 5′UTR in Xenopus laevis oocytes.(A) Schematic representation of the experimental procedure. (B) Capped and polyadenylated RNA corresponding to the dl ?EMCV [10], [25], dl HIV-1 IRES [10], [19], [20], dl HBX2 (pLAI), or the different dl VAR 5′UTR (VAR 1, VAR 2, VAR 3, VAR 4, VAR 12) vectors (6.25 ng) were microinjected (50 nl final volume) into Xenopus laevis oocytes as previously described [19], [20], [23]. Oocytes were harvested 24 hr after microinjection, processed, and Renilla luciferase (RLuc) and Firefly luciferase (FLuc) activities were determined as described in Materials and Methods. Data are expressed as relative luciferase activities, the RLuc and FLuc activities of the dl HIV-1 IRES, the control bicistronic RNA harboring the 5′UTR of pNL4.3 in the intercistronic region between RLuc and FLuc [10], was arbitrarily set at 1. Each value is the mean +/- SEM from at least 3 oocytes.
	Figure 4. Analysis of a promoterless bicistronic construct containing the HIV-1 5′UTR sequences recovered from clinical samples.(A) Schematic representation of the bicistronic constructs. The SV40 promoter from dl ΔEMCV (lane 4), dl HIV-1 IRES, or dl VAR was removed to generate the equivalent promoterless (ΔSV40) vectors. (B) HeLa cells were transfected with DNA (200 ng) corresponding to the vectors depicted in (A) as previously described [23]. Total DNA was extracted from transfected cells and the presence of the transfected plasmids was confirmed by PCR (upper panel). The dl HIV-1 IRES plasmid (100 ng) was used as an amplification control. Total RNA was extracted from transfected cells and the presence of transcripts for the ΔSV40-dl ΔEMCV, ΔSV40-dl HIV-1 IRES, or the ΔSV40-dl VAR plasmids was evaluated by a one step RT-PCR designed to detect the bicistronic RNA (depicted in Fig. 2B) [23]. In vitro transcribed RNA (100 ng) generated from plasmids dl HIV-1 IRES (lane 2) were used as amplification controls. (C) HeLa cells were transfected with either the 200 ng of SV40 or ΔSV40 version of dl ΔEMCV, dl HIV-1 IRES, or the different dl VAR plasmids as previously described [23]. Cells were processed and RLuc and FLuc activities were measured. For each data point the [RLuc/(total protein)] (left panel) and the [FLuc/(total protein)] (right panel) for the SV40 positive plasmids was arbitrary set to 100%. Values are the means +/- SD from three independent experiments.
	Figure 5. Secondary structure model of the 5′UTR of selected HIV-1 VAR sequences.The HIV-1 5′leader recovered from the dl VAR constructs (followed by 58 nucleotides of fLuc gene) was probed using Dimethyl Sulfate (DMS), N-Cyclohexyl-N0- [N-Methylmorpholino)-ethyl]-Carbodiimide-4-Toluolsulfonate (CMCT) and RNAse V1 as previously described [20]. (A) Secondary structure model of the control (pNL4.3) HIV-1 5′UTR according to DMS, CMCT and RNAse V1 structure probing [6], a key indicating the respective reactivity to the different probes is provided (box). Data included in the figure have been extracted from reference [20]. (B) Typical examples of DMS probing. The HIV-1 5′leader was probed using (+) DMS. Reverse transcription (RT) products were separated on a 8% gel as previously described [12], [20], [48]. Sequencing lanes were also included. Note that DMS induces a premature RT stop one nucleotide before the hit. Therefore the DMS induced stops migrate faster than the corresponding sequence product [12], [20], [48]. The RT pattern of the modified RNA was compared to the profile obtained with an unmodified RNA. Some hits are indicated in the figure. The asterisks on the gel denote the FLuc initiation codon (AUG). Results for VAR 1 (C), VAR 2 (D), VAR 3 (E), and VAR 4 (F) were fitted in a model of the HIV-1 5′ leader [6] as previously described [20]. Numbering in A-F is with respect to clone pNL4.3, here considered the wt sequence. Insertions are indicated in red as independent numbers (iN, were N is the number). Nucleotide changes with respect to clone pNL4.3 are indicated in green. The nucleotide located before a deletion is highlighted in yellow, in this case numbering with respect to pNL4.3 is not altered.
	Figure 6. The hnRNPA1 protein enhances translation from the HIV-1 5′UTR.HeLa cells were transfected with either the plasmid expressing hnRNPA1 or the equivalent empty vector in combination with dl HIV-1 IRES or the different dl VAR vectors. Cells were processed and RLuc and FLuc activities were measured. The FLuc/RLuc ratio for each individual bicistronic vector in presence of the empty vector was arbitrary set as the base line. The increase in FLuc/RLuc ratio, which reflects the increase of HIV-1 IRES activity, in presence of the plasmid expressing hnRNPA1 for each individual bicistronic vector is graphed as percentage (%) of stimulation. Values are the means +/- SEM from three independent experiments.

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