The frameshift stimulatory signal of human immunodeficiency virus type 1 group O is a pseudoknot

Abstract

Human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshift to produce Gag-Pol, the precursor of its enzymatic activities. This frameshift occurs at a slippery sequence on the viral messenger RNA and is stimulated by a specific structure, downstream of the shift site. While in group M, the most abundant HIV-1 group, the frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group O. The mutagenesis and probing studies coupled to an in silico analysis show that group O pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2nt (loop 1) and 20nt (loop 2). Mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the frameshift efficiency to near wild-type level. The difference between the frameshift stimulatory signal of group O and group M supports the hypothesis that these groups originate from a different monkey to human transmission.

Figure 1

Predicted structure for the frameshift region of subtype MVP5180 of HIV-1 group O. (a) The slippery sequence (underlined) is followed by an 8-nt spacer and an 8-bp stem, capped by a 10-nt loop. Eight nucleotides of this loop could base-pair with a complementary region downstream of the stem-loop (proposed pairings are represented by broken lines). Such an interaction results in a pseudoknot with an 8-nt stem 1, a 2-nt loop 1, an 8-nt stem 2 and a 20-nt loop 2. (b) Structure of the frameshift region of subtype B of HIV-1 group M, as determined by Dulude et al.¹⁴

Figure 2

Description of the luciferase expression vectors used for the study of the programmed −1 ribosomal frameshift of HIV-1 group O in vitro and in cultured cells. (a) The HIV-1 frameshift region of subtype MVP5180 of group O was inserted upstream of the coding sequence of the luciferase reporter gene, generating construct pHIV/O-87-LUC. The slippery site is UUUUUUA (underlined). All mutants of subtype MVP5180 were cloned by inserting between the KpnI-BamHI sites of the vector, the PCR product bearing the mutation investigated. For subtype ANT70 of group O, the corresponding vector was constructed by exchanging the Eco47III-BamHI fragment 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 was added immediately after the slippery sequence (at position 25) 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 constructs used in this study. Nucleotides substituted or deleted compared to subtype MVP5180 of group O are underlined or represented by broken lines, respectively.

Figure 3

Effect of different mutations in the frameshift region of subtype MVP5180 of HIV-1 group O on the frameshift efficiency. (a) A series of mutations were made within the frameshift region of pHIV/O-87-LUC (the dots correspond to the BamHI linker connecting the frameshift region to the luciferase coding sequence): a slippery site mutant, pHIV/O-k/o-LUC, where the slippery sequence (underlined) is mutated (bases that are changed are in capital letters); deletion mutant pHIV/O-DSL-LUC, where the region encompassing stem 1 and its capping loop is deleted (deletion of bases 33 to 60); deletion mutant pHIV/O-60-LUC, where the region 3′ to stem 1 is deleted; substitution mutants where the 3′ strand of stem 1 is altered, impairing formation of stem 1 (pHIV/O-1.2-LUC), and where the 3′ and 5′ strands of stem 1 are simultaneously altered, allowing re-formation of stem 1 (pHIV/O-1.12-LUC). (b) Frameshift efficiency in vitro and in cultured cells for the pHIV/O-LUC constructs described above. In vitro translation experiments were made in 25 μl of RRL with 0.2 μg of mRNAs transcribed from the StuI-digested pHIV/O-LUC constructs. Assays in cultured cells were made by co-transfecting 293T cells with 3 μg of pHIV/O-LUC and 1.25 μg of pcDNA3.1/Hygro(+)/lacZ. Frameshift efficiencies were calculated as described in the text. Each value represents the mean±standard error of five to six independent experiments. The bars indicate the standard error on the mean.

Figure 4

Effect on the frameshift efficiency of mutations impairing formation of stem 2 of the pseudoknot of subtype MVP5180 of HIV-1 group O. (a) Description of mutations made within the gag/pol frameshift region of pHIV/O-87-LUC (the dots correspond to the BamHI linker connecting the frameshift region to the luciferase coding sequence). Mutations were introduced either in the loop capping stem 1 or in the complementary downstream region, impairing formation of stem 2 (pHIV/O-2.1-LUC and pHIV/O-2.2-LUC, respectively), or allowing re-formation of this stem (pHIV/O-2.12-LUC). Subtype ANT70 of group O, where sequence differences compared to subtype MVP5180 impair formation of stem 2, was also assessed. (b) Frameshift efficiency in vitro and in cultured cells with the HIV/O-LUC constructs described above. Assays were as described in the legend to Figure 3.

Figure 5

Probing of the structure proposed for the frameshift stimulatory signal of subtype MVP5180 of HIV-1 group O. (a) Structure probing of the frameshift stimulatory signal by RNase V₁ attack. An RNA transcript encompassing the gag/pol frameshift region was 5′ end-labeled with γ-³²P and digested with RNase V₁. Digestion products were analyzed on a 20% (left) and a 10% (right) acrylamide 7 M urea gel. The sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH⁻). The nucleotides that were cleaved were identified by the absence of cleavage in the untreated control lane (0). The amount of units of enzyme added to each reaction is also indicated. (b) Summary of the RNase V₁ attacks in the pseudoknot structure of the frameshift stimulatory signal of subtype MVP5180 of HIV-1 group O. The sensitivity of nucleotides to RNase V₁ is shown by arrows of different size, where the size is approximately proportional to the intensity of the cleavage at that site. Bases 1 to 62 originate from subtype MVP5180, while bases 63 to 66 (in gray) originate from the vector.

Figure 6

Stereo view of the computer modeled structure for the pseudoknot of subtype MVP5180 of HIV-1 group O. Stems (S1 and S2) and loops (L1 and L2) are represented in different colors. (a) Structure of the MVP5180 pseudoknot, with a putative sub-stem (SS) in loop 2 (see the text). Nucleotides in the loop of this sub-stem were not included in our modeling. (b) Schematic representation of the pseudoknot.