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, 14 (7), 1443-55

In Vitro Expression of the HIV-2 Genomic RNA Is Controlled by Three Distinct Internal Ribosome Entry Segments That Are Regulated by the HIV Protease and the Gag Polyprotein

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In Vitro Expression of the HIV-2 Genomic RNA Is Controlled by Three Distinct Internal Ribosome Entry Segments That Are Regulated by the HIV Protease and the Gag Polyprotein

Emiliano P Ricci et al. RNA.

Abstract

The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease to show that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production and also on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.

Figures

FIGURE 1.
FIGURE 1.
Mapping HIV-2 IRES activity. (A) Schematic representation of the bicistronic constructs (pBi) used in this study. Different regions of the HIV-2 genomic RNA were inserted in the intercistronic spacer of the Neomycin-LacZ bicistronic vector. (B) In vitro translation in the rabbit reticulocyte lysate of the uncapped bicistronic RNA constructs (200 ng/10 μL) as indicated on top of the figure. Translation products were then resolved on a 15% SDS-PAGE and subjected to autoradiography. Results are representative of at least three independent experiments.
FIGURE 2.
FIGURE 2.
IRES activity in HeLa cells. (A) Schematic representation of the dual luciferase bicistronic constructs (pFR) used in this study. Different regions of the HIV-2 genomic RNA were inserted in the intercistronic spacer of the firefly-Renilla bicistronic vector (see Materials and Methods). (B) Western blot analysis of eIF4G from HeLa cells transfected with an RNA coding for the 2A protease from poliovirus. (C) Transfection of capped and polyadenylated bicistronic RNA constructs (0.156 pmol) in HeLa cells previously transfected (p2A) or not (Mock) with an RNA coding for the p2A protease. Luciferase activities were measured 3 h post-transfection. R/F ratios were calculated and normalized to the value of the pFR-NoIRES construct. Error bars represent the standard deviation obtained from three independent experiments.
FIGURE 3.
FIGURE 3.
Cleavage of eIF4G stimulates HIV-2 translation. A RRL under full translation conditions was preincubated for 10 min without (lanes 1,4,7,10,13,16) or with 0.3 μL (lanes 2,5,8,11,14,17) or 0.7 μL (lanes 3,6,9,12,15,18) of in vitro produced FMDV L-protease. Globin-LacZ (15 ng/10 μL), EMCV-LacZ (184 ng/10 μL), T7-WT (200 ng/10 μL), and T7-AUG1 (35 ng/10 μL) were translated as indicated on the figure and the resulting products resolved on a 13% SDS-PAGE and submitted to autoradiography. The relative intensities of the bands were quantified using a storm 850 phosphoimager and expressed as arbitrary units presented at the bottom of each panel. T7-WT and T7-AUG1 overall translation was quantified by the addition of the activities of each of the Gag isoforms. Results are representative of at least three independent experiments.
FIGURE 4.
FIGURE 4.
Translation of the HIV-2 T7-WT RNA in a competitive cap and poly-A dependent RRL. Translation of increasing amounts (50, 100, and 200 ng) of capped (lanes 1–3) and uncapped (lanes 4–6) poly-adenylated T7-WT RNA in the untreated RRL containing endogenous Globin and lipoxygenase mRNAs as indicated on the figure. Translation of 200 ng of capped and poly-adenylated T7-WT RNA following preincubation of the untreated RRL for 10 min without (lane 7) or with 0.3 μL (lane 8), or 0.7 μL (lane 9), or with 1 μL (lane 10) of in vitro produced FMDV L-protease. Translation products were resolved on a 13% SDS-PAGE and submitted to autoradiography. Results are representative of at least three independent experiments.
FIGURE 5.
FIGURE 5.
Three independent IRES control p57, p50, and p44 protein expression. Increasing concentrations (lane 1: 0 μM, lane 2: 50 μM, lane 3: 100 μM, and lane 4: 250 μM) of antisense 2′-O-methyloligoribonucleotides directed against the 5′-UTR of globin-LacZ (lanes 1–4), the primary binding site (PBS) of HIV-2 (lanes 5–8), the region encompassing AUG1 (9–12), or the region encompassing AUG2 (lanes 13–16) were hybridized to capped T7-WT RNA (200 ng/10 μL) and the resulting oligo–RNA complex was translated in the RRL. Translation products were resolved on a 13% SDS-PAGE and submitted to autoradiography. The relative intensities of the bands were quantified using a storm 850 phosphoimager and expressed as arbitrary units presented on the right-hand side of each panel. The position of translation products is indicated on the figure. Results are representative of at least three independent experiments.
FIGURE 6.
FIGURE 6.
Addition of the recombinant HIV-2 protease modifies AUG codon selection. A RRL under full translation conditions was preincubated for 1 h without (lanes 1,4,7,10,13,16,19) or with 3 ng/μL (lanes 2,5,8,11,14,17,20), or 5 ng/μL (lanes 3,6,9,12,15,18,21) of recombinant HIV-2 protease prior to addition of Palinavir (10 μM). The following transcripts: Globin-LacZ (15 ng/10 μL), EMCV-LacZ (184 ng/10 μL), HCV NS (100 ng/10 μL), T7-WT (200 ng/10 μL), and T7-AUG1 (35 ng/10 μL) were then translated as indicated on the figure and the resulting products resolved on a 10% SDS-PAGE and submitted to autoradiography. The relative intensities of the bands were quantified using a storm 850 phosphoimager and expressed as arbitrary units presented at the bottom of each panel. T7-WT and T7-AUG1 overall translation was quantified by the addition of the activities of each of the Gag isoforms. Results are representative of at least three independent experiments.
FIGURE 7.
FIGURE 7.
The Gag recombinant protein exerts a regulatory effect on the translation of the HIV-2 genomic RNA. Capped globin-LacZ (15 ng/10 μL, lanes 1–9), capped EMCV-LacZ (184 ng/10 μL, lanes 10–18), capped T7-WT (200 ng/10 μL, lanes 19–27), capped T7-AUG1 (35 ng/10 μL, lanes 28–36), and capped Globin∷Gag (35 ng/10 μL, lanes 37–45) RNAs were preincubated for 10 min at 30°C in the presence of increasing amounts of recombinant HIV-1 Gag protein ranging from 0 to 82 Gag molecules per RNA as indicated on the figure. The resulting Gag-RNAs complexes were then translated in the RRL for 45 min and the resulting products were resolved on a 13% SDS-PAGE and submitted to autoradiography. The relative intensities of the bands were quantified using a storm 850 phosphoimager and presented at the bottom of each panel.
FIGURE 8.
FIGURE 8.
The 5′-UTR of the HIV-2 genomic RNA act as a dock for the Gag polyprotein thus having an inhibitory effect on its own translation. RRL under full translational conditions was incubated in the absence (lanes 1,7) or presence of a large excess of recombinant Gag protein (all other lanes, 82:1 protein to RNA ratio). Uncapped T7-WT (15 ng/μL) and T7-AUG1 (5 ng/μL) RNAs were translated in the absence (lanes 2,8) or presence of the HIV-2 5′-UTR added in trans (lanes 5,6,11,12) or an unspecific β-Gal RNA (lanes 3,4,9,10). Samples were processed on a 12% SDS-PAGE and submitted to autoradiography.

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