Internal RNA 2'-O-methylation on the HIV-1 genome impairs reverse transcription

Abstract

Viral RNA genomes are modified by epitranscriptomic marks, including 2'-O-methylation that is added by cellular or viral methyltransferases. 2'-O-Methylation modulates RNA structure, function and discrimination between self- and non-self-RNA by innate immune sensors such as RIG-I-like receptors. This is illustrated by human immunodeficiency virus type-1 (HIV-1) that decorates its RNA genome through hijacking the cellular FTSJ3 2'-O-methyltransferase, thereby limiting immune sensing and interferon production. However, the impact of such an RNA modification during viral genome replication is poorly understood. Here we show by performing endogenous reverse transcription on methylated or hypomethylated HIV-1 particles, that 2'-O-methylation negatively affects HIV-1 reverse transcriptase activity. Biochemical assays confirm that RNA 2'-O-methylation impedes reverse transcriptase activity, especially at low dNTP concentrations reflecting those in quiescent cells, by reducing nucleotide incorporation efficiency and impairing translocation. Mutagenesis highlights K70 as a critical amino acid for the reverse transcriptase to bypass 2'-O-methylation. Hence, the observed antiviral effect due to viral RNA 2'-O-methylation antagonizes the FTSJ3-mediated proviral effects, suggesting the fine-tuning of RNA methylation during viral replication.

Graphical Abstract

Figure 1.

2′-O-Methylated HIV-1 genomes are less prone to replication in cells. (A) Total RNA was extracted from HEK293T WT or FSTJ3 KO cells and the relative expression of FTSJ3 transcripts was quantified by RT–qPCR. Data represent the mean ± standard deviation (SD) of duplicates. (B) WT HIV-1 particles produced in HEK293T WT or FSTJ3 KO cells were submitted to ERT for 5 or 10 h, as indicated. The amount of reverse transcriptase products was estimated by qPCR. The cDNA copy numbers per ng of virus are shown (n = 12 from four independent experiments), with the bars showing the mean ± SD. **P < 0.01, ****P < 0.0001, as determined by Student's t-test. (C) HEK293T cells treated or not with 1 mM hydroxyurea (HU) for 24 h, before the cell cycle profiles were assessed by PI labeling. (D) HEK293T cells treated or not with HU were transduced with VSV-G pseudotyped HIV-1 particles produced in WT or FSTJ3 KO HEK293T cells, as indicated. At 6 h post-infection, total cellular DNA was extracted, and HIV-1 reverse transcriptase products were quantified by qPCR. Data represent the mean ± SD of three independent experiments performed in duplicate. ****P < 0.0001, as determined by Student's t-test. (E) Jurkat cells treated or not with 1 mM HU for 24 h, before the cell cycle profiles were assessed by PI labeling. (F) Jurkat cells treated or not with HU were transduced with HIV-1 particles produced in WT or FSTJ3 KO HEK293T cells, as indicated. At 6 h post-infection, total cellular RNA was extracted, and HIV-1 reverse transcriptase products were quantified by qPCR. Data represent the mean ± SD of three independent experiments performed in duplicate. ****P < 0.0001, as determined by Student's t-test.

Figure 2.

Primer extension assays on a 2′-O-methylated RNA template reveal a pause during reverse transcription at low dNTP concentration. (A) Sequences of the two DNA/RNA primer/template (P/T) duplexes used for primer extension assays. The DNA primers are both 10-mer and Cy5-labeled. The RNA templates have identical sequences, with RNA-A_m27 being 2′-O-methylated on the 27th nucleotide defined from the 3′ end. (B) Primer extension assays were carried out on both templates shown in (A) with [RT_WT] = 0.08 μM, [P/T] = 0.02 μM, [dNTP] = 0.5 μM. Reverse transcriptase (RT) is incubated for 5 min at 37°C with either P/T duplex. The reaction starts by the addition of dNTP, and is quenched at several time points (0, 0.5, 1, 5, 10, 20 and 50 min, as indicated at the bottom of the gel) in a 10 mM formamide–EDTA buffer. Samples are loaded on a denaturing PAGE gel (20% acrylamide, 7 M urea) and detected by a fluorescence imager (Amersham Typhoon). A highly contrasted lane along with the elongated DNA sequence, corresponding to the primer extension assay on RNA-A_m27 after 50 min, juxtaposes the gels. (C and D) Quantification of initial DNA (10-mer), intermediate DNA products (26-mer) and full-length DNA (40-mer) resulting from primer extension assays for RNA control and RNA-A_m27, respectively. Each data point represents the mean ± SD of three independent experiments.

Figure 3.

Reverse transcriptase (RT) pause depends on the dNTP concentration and the 2′-O-methylated nucleic acid base. (A–C) Primer extension assays were carried out on primer/template RNA-A_m27 shown in Figure 2A. (A) Primer extension assays were performed in the following conditions: [RT_WT] = 0.8 μM, [P/T] = 0.2 μM, four dNTP concentrations as indicated (0.5, 1, 5 and 50 μM), and quenched at different times (0, 0.5, 1, 5, 10, 20 and 50 min). The samples were separated on a denaturing PAGE gel (20% acrylamide, 7 M urea), and visualized using a fluorescence imager (Amersham Typhoon). (B) Quantification of the 26-mer intermediate band of the primer extension assays shown in (A). Each data point represents the mean ± SD of three independent experiments. (C) Quantification of the 26-mer intermediate band of primer extension assays performed with [RT_WT] = 0.08 μM, [P/T] = 0.02 μM and [dNTP] = 1 μM, plus an excess concentration of another dNTP as indicated, resulting in a 20 μM concentration of the respective dNTP. Each data point represents the mean ± SD of three independent experiments. (D) Quantification of the 26-mer intermediate band of primer extension assays performed with [RT_WT] = 0.08 μM, [P/T] = 0.02 μM and [dNTP] = 1 μM on the previously used DNA primer (A) annealed to four different RNA templates (see Supplementary Table S2) where the 27th position site is either a G_m, A_m, U_m or C_m. Each data point is the mean ± SD of three independent experiments.

Figure 4.

The chemistry step opposite a 2′-O-methylated A site represents a limiting step in reverse transcription. (A) Two duplexes of DNA/RNA primer/templates (P/T) used for FP and primer extension assays. DNA primers are 17-mer Cy5-labeled oligonucleotides. RNA templates have the same sequence, except that midRNA-A_m27 is 2′-O-methylated on the 18th nucleotide, as defined from the 3′ end. (B) Fluorescence polarization with 0.0125 μM of both P/T pairs and a 0.00061–10 μM range of reverse transcriptase concentrations. The data were fitted with a quadratic form binding equation curve, yielding similar K_d values of 16 ± 3 nM for the midRNA-A_m27 template and 13 ± 3 nM for the midRNA-control template. Each data point represents the mean ± SD of three independent experiments. (C) The K_m of dTTP for the P/T·RT complex was assessed by performing primer extension assay, mixing the reverse transcriptase, either of the P/T duplexes shown in (A) and a range of dTTP concentrations (0.01, 0.1, 0.2, 0.5, 1, 5, 10, 15 and 20 μM), prior to stopping the reaction at different times (0, 0.5, 1, 2, 5, 10 and 20 min) in 10 mM formamide–EDTA buffer. The samples are analyzed using denaturing PAGE (20% acrylamide, 7 M urea), and scanned using a fluorescence imager (Amersham Typhoon). The elongated DNA products were quantified and plotted against time. The slope coefficient allowed determination of the initial speed (V_i, pmol/min) by linear regression in the linear fraction of the curve. The V_i values were plotted against the dTTP concentration prior to a Michaelis–Menten fit, allowing the determination of K_m and V_max. This figure shows the typical results of one replicate, but this experiment was done as a duplicate, yielding similar results (see Table 1 for the data of the duplicate). (D and E) The K_m (D) or the V_max (E) of dTTP were extrapolated from two independent experiments performed on each template. ‘midRNA-control₁’ and ‘midRNA-Am27₁’ are the K_m or V_max ± standard error (SE) of the fit of replicate 1, and ‘midRNA-control₂’ and ‘midRNA-Am27₂’ are the K_m or V_max ± SE of the fit of replicate 2. Both K_m values or V_max values were also pooled together to obtain the mean ± SEM of the K_m or V_max for the duplicate (referred to as ‘midRNA-control₁₊₂’ and ‘midRNA-Am27₁₊₂’). Data are differentiated by colors, as indicated next to (E).

Figure 5.

2′-O-Methylation promotes excision of the DNA primer. (A and B) Time courses of the build-up of excision products at 0, 1, 5, 10, 20, 50 and 100 μM PPi (light to dark blue or red) for the control (A) and A_m27 (B) template RNA strands. The data show burst kinetics that suggest that a portion of the P/T pre-exists in the backtracked state and is poised for rapid excision, while the remainder of the P/T is excised on a slower time scale limited by rebinding or backtracking rates. The A_m27 RNA results in a larger burst amplitude and an ∼3-fold faster continued excision rate that are both indicative of more efficient backtracking with the 2′-O-methylated RNA base. (C) Concentration dependence curves show different maximum burst amplitudes of 67 ± 6 nM for control RNA (blue) and 97 ± 8 nM for A_m27 RNA (red), but no change in the midpoint PPi concentrations which is 5.4 ± 2 μM.

Figure 6.

Model of the structural features of a 2′-O-methylation addition on a DNA/RNA–reverse transcriptase competent complex. (A) A NUCPLOT analysis was performed on PDB 4pqu, a structure of a reverse transcriptase bound to a DNA/RNA primer/template duplex, with an incoming dATP in the polymerase active site. This analysis shows the interactions between the p66 subunit (residue number and atom name, chain A) and nucleic acids (chains T and P for RNA and DNA, respectively). Interactions with water molecules are not shown, −1 elongated site, +1 and +2 sites for elongation are identified. (B–D) Structure of the HIV reverse transcriptase–P/T complex (PDB 4pqu) with 2′-O-methyl modeled in the +1 and +2 template strand positions. Reverse transcriptase (gray), DNA (tan), RNA (yellow), incoming dATP (green), 2′-O-methyl (magenta, half-sized spheres). (B) Overview of the active site with +1 position (N-site) base shown in cyan and base-paired to a dNTP (green) bound in the active site. 2′-O-Methyl was modeled onto the RNA at the +1 and +2 position based on superpositioning of ribose groups from PDB 310d. (C) Detailed view showing that a 2′-O-methyl in the +1 position (cyan) will be placed in a polar environment with a significant steric clash with G152 carbonyl and unfavorable backbone interactions with V75–F77. In contrast, a 2′-O-methyl in the +2 position (dark cyan) will be in a more hydrophobic environment involving C_β of D76 and the aromatic rings of F61 and W24. (D) Amino acids targeted for mutagenesis are shown in cyan and colored by atom in the vicinity of the reverse transcriptase active site.

Figure 7.

Endogenous reverse transcription in cell-free particles of mutant HIV-1 containing WT or hypomethylated genomes. (A and B) HIV-1 WT or mutant reverse transcriptases were produced in HEK293T WT or FSTJ3 KO cells and submitted to ERT for 10 h. The amount of reverse transcriptase products was estimated by qPCR. Results are represented as mean ± SD and shown as raw values (A) or as ratios of reverse transcriptase products in viruses produced in HEK293T KO versus the WT (B). **P < 0.01 and ****P < 0.0001 as determined by Student's t-tests.