Efficiency of a programmed -1 ribosomal frameshift in the different subtypes of the human immunodeficiency virus type 1 group M

Abstract

The synthesis of the Gag-Pol polyprotein, the precursor of the enzymes of the human immunodeficiency virus type 1 (HIV-1), requires a programmed -1 ribosomal frameshift. This frameshift has been investigated so far only for subtype B of HIV-1 group M. In this subtype, the frameshift stimulatory signal was found to be a two-stem helix, in which a three-purine bulge interrupts the two stems. In this study, using a luciferase reporter system, we compare, for the first time, the frameshift efficiency of all the subtypes of group M. Mutants of subtype B, including a natural variant were also investigated. Our results with mutants of subtype B confirm that the bulge and the lower stem of the frameshift stimulatory signal contribute to the frameshift in addition to the upper stem-loop considered previously as the sole participant. Our results also show that the frameshift stimulatory signal of all of the other subtypes of group M can be folded into the same structure as in subtype B, despite sequence variations. Moreover, the frameshift efficiency of these subtypes, when assessed in cultured cells, falls within a narrow window (the maximal deviation from the mean value calculated from the experimental values of all the subtypes being approximately 35%), although the predicted thermodynamic stability of the frameshift stimulatory signal differs between the subtypes (from -17.2 kcal/mole to -26.2 kcal/mole). The fact that the frameshift efficiencies fall within a narrow range for all of the subtypes of HIV-1 group M stresses the potential of the frameshift event as an antiviral target.

FIGURE 1.

Vectors expressing luciferase used to study the programmed −1 ribosomal frameshift of HIV-1 group M in vitro and in cultured cells. (A) The frameshift region of subtype B was inserted upstream of the coding sequence of the luciferase reporter gene, which is under control of a CMV and a T7 promoter, generating pLUC/HIV/B (−1). The classic structure of the frameshift stimulatory signal is represented here. For the study of all subtypes of group M and mutants of subtype B, the corresponding vectors were constructed by exchanging the Eco47III–BamHI fragment of pLUC/HIV/B (-1) with an appropriate oligonucleotide cassette. For the (-1) constructs, the luciferase sequence is in the −1 reading frame relative to the AUG initiation codon, so that a −1 frameshift is required to produce luciferase. An adenine nucleotide was added between the frameshift region and luciferase sequence for the (0) constructs, so that luciferase is expressed by ribosomes that do not shift the reading frame. (B) Sequences of the frameshift region of all of the constructs used in this study. Nucleotides modified or deleted relative to subtype B are underlined or represented by broken lines, respectively.

FIGURE 2.

Relative frameshift efficiency of mutants of subtype B of HIV-1 group M. (A) Secondary structure of the frameshift stimulatory signal for subtype B, subtype B short, subtype B bulge subs, and subtype B*, a natural variant that has a deletion of three nucleotides in the frameshift stimulatory signal. The stimulatory signal is represented with the extended bulged stem–loop structure found for HIV-1 subtype B (Dulude et al. 2002). Circles indicate nucleotides that differ from subtype B. The thermodynamic stability, ΔG° (kcal/mole), of the frameshift stimulatory signal of the different mutants of subtype B is indicated. The predicted ΔG° were calculated with the mfold program of M. Zuker (Mathews et al. 1999) for the classic stem–loop structure (upper stem) and the extended bulged stem–loop structure (upper stem + lower stem). The complete frameshift region with the slippery sequence underlined is represented for subtype B, whereas only the frameshift stimulatory signal is shown for the mutants. (B) Relative frameshift efficiency of mutants of subtype B. In vitro studies in RRL and assays in cultured cells (293T) were performed as described in the text. For assays in cultured cells, the experimental values were normalized for variations in transfection efficiency by cotransfecting the various constructs used for measuring the frameshift efficiency with pcDNA3.1/Hygro(+)/lacZ and monitoring the β-galactosidase activity. The frameshift efficiency of subtype B is arbitrarily set at 100%. The values of the frameshift efficiency are the means of at least five independent experiments, with bars representing the standard error on the means.

FIGURE 3.

Secondary structure of the frameshift stimulatory signal of all of the subtypes of HIV-1 group M. The frameshift stimulatory region is folded according to the extended bulged stem–loop structure found for HIV-1 subtype B stimulator (Dulude et al. 2002). Circles indicate nucleotides that differ from subtype B. The arrows point to noncanonical base pairs and mismatches. The thermodynamic stability, ΔG° (kcal/mole), of the frameshift stimulatory signal of the different subtypes is indicated. The predicted ΔG° were calculated as described in the legend to Figure 2 ▶.

FIGURE 4.

Relative frameshift efficiency of all subtypes of HIV-1 group M. In vitro studies in RRL and assays in cultured cells (293T) were performed as indicated in Figure 2 ▶. The mean value of the frameshift efficiency, calculated from the values obtained for all of the subtypes (1.9% in cultured cells and 6.0% in vitro), is arbitrarily set at 100%. The frameshift efficiency of subtype B* has been added to the figure to facilitate comparison with the different subtypes. The values of the frameshift efficiency are the means of at least five independent experiments, with bars representing the standard error on the means.