G-quadruplex-forming small RNA inhibits coronavirus and influenza A virus replication

Abstract

Future pandemic threats may be caused by novel coronaviruses and influenza A viruses. Here we show that when directly added to a cell culture, 12mer guanine RNA (G12) and its phosphorothioate-linked derivatives (G12(S)), rapidly entered cytoplasm and suppressed the propagation of human coronaviruses and influenza A viruses to between 1/100 and nearly 1/1000 of normal virus infectivity without cellular toxicity and induction of innate immunity. Moreover, G12(S) alleviated the weight loss caused by coronavirus infection in mice. G12(S) might exhibit a stable G-tetrad with left-handed parallel-stranded G-quadruplex, and inhibit the replication process by impeding interaction between viral nucleoproteins and viral RNA in the cytoplasm. Unlike previous antiviral strategies that target the G-quadruplexes of the viral genome, we now show that excess exogenous G-quadruplex-forming small RNA displaces genomic RNA from ribonucleoprotein, effectively inhibiting viral replication. The approach has the potential to facilitate the creation of versatile middle-molecule antivirals featuring lipid nanoparticle-free delivery.

Fig. 1. Uptake, intracellular distribution of G12 and G12(S) added directly to the culture medium.

Images of the TAMRA-G12, TAMRA-G12(S), and nuclei (Hoechst 33342) in A549 cells were observed at the times indicated below and are shown in green (TAMRA) and blue (Hoechst 33342) pseudocolors, respectively (a–e). a Cellular uptake of TAMRA-G12 (2 μM, 30 min). b Uptake of TAMRA-G12(S) (0.2 μM) in the absence or presence of a 10-fold concentration of unlabeled G12(S) (2 μM) for 1 h. c Time-dependent incorporation of TAMRA-G12(S) signals from 0 to 15 min after the addition of 2 μM TAMRA-G12(S). d FRAP analysis of TAMRA-G12(S). Fluorescence was bleached for 5 min at 100% laser power. The photobleached area is marked with a white circle in the leftmost image. Images were acquired over time for the times (min) shown in the figure. e Distribution of TAMRA-G12(S) (green) and ER-Tracker Red (red). Cells were treated with 2 μM TAMRA-G12(S) in the medium for 1 h and further incubated with 1 μM ER-Tracker Red and NucBlue (Hoechst 33342). Scale bars are shown in (a–e). f Cytotoxicity of G12(S) on A549. The cell viabilities were examined after 24 h of culture in the presence of 0–10 μM G12(S) using AlamerBlue. One-way analysis of variance (ANOVA) was used for the statistical analysis (n = 3, P-values = 0.3582, F-value = 1.230). There is no significant difference between the combinations. g Effect of G12(S) on protein synthesis in A549 were determined using the puromycin uptake assay SuNSET method. Cells were cultured as a condition of the virus infection in the presence of 1 and 2 μM G12(S) for 24 h. Cycloheximide (10 μg/mL CHM) instead of G12(S) was used to inhibit protein synthesis as a control. See Supplementary Fig. 1f for western blotting data for the SuNSET. Reporter gene assays for IFN-β promoter (h) and a promoter containing the NFκB binding sites (i) in HEK293T cells. The values shown are relative values when the control (without G12(S) and poly I:C) is set as “1”. The reporter genes activated by polyI:C were not induced by 2 μM G12(S), but rather suppressed. Statistical analysis of the significance for (g–i) were carried out by One-Way ANOVA (g n = 3, F(3,8) = 43.26, P < 0.0001; h n = 3, F(3, 8) = 67.30, P < 0.0001; i n = 3, F (3, 8) = 290.1, P < 0.0001), and Dunnett’s multiple comparison test (g) or Tukey’s multiple comparison test (h, i). Data are shown by Box-and-whisker plot are used with P-values for the multiple comparison (f–i).

Fig. 2. Effect of G12(S) on β coronavirus OC43 replication in infected human cells and mice.

OC43 NP levels in HCT-8 cells infected with OC43 (multicity of infection (MOI) = 0.01, n = 3) in the absence (a) and presence of 2 μM G12(S) in the medium (b). Cell lysates were prepared at 2, 3, 4, and 5 days post-infection (dpi) in the absence (a) and presence of 2 μM G12(S) in the medium (b), and determined by western blotting with anti-OC43 NP and anti-actin antibodies. c Changes in NP/actin ratio (n = 3, normalized with the ratio of (a) at 2 dpi as 1) without (−, from a) and with (+, from b) G12(S). d OC43 RNA levels in OC43-infected (MOI = 0.01) cells (left) and culture medium (right) in the absence (control) and in the presence of 2 μM G12(S). e Effect of G12(S) on OC43 virus titers (pfu) of the medium. f OC43 RNA levels in the culture medium. Statistical analyses of the significance of G12(S) effect were performed by Two-Way ANOVA and data are shown by box-and-whisker plots including all data plots (c–f) (c n = 3, F(3,8) = 25.28, P = 0.0002; d left, n = 3, F(1,4) = 250.8, P < 0.0001; d right, n = 3, F(1,4) = 1800, P < 0.0001; e n = 3, F(1,4) = 5.245, P = 0.0838; f n = 3, F(1,4) = 77.53, P = 0.0009). Sidak’s multiple comparison test was used and the P-values are indicated. g Effect of 1 and 2 μM G12(S) on the CPE of OC43 (MOI = 0.1)-infected MRC5 cells (3 dpi). h OC43 infection in nasal cavities and olfactory bulbs of newborn mice. Immunofluorescence images of OC43 NP (yellow) and nucleus (DAPI, blue) are shown. Scale bars indicate 1 mm (i) and 20 μm (iii, iv) lengths. See Supplementary Fig. 3 for entire images. i, j Prevention of mice toxicity due to OC43 mouse infection by G12(S). OC43 infection and single-dose (i, n = 22) and three-dose (j, n = 13) schedules of G12(S) are shown. The survival rate is shown as Kaplan–Meier curves. P-values were determined by Logrank test.

Fig. 3. Effect of G12(S) on the propagation of 229E and SARS-CoV-2.

a 229E NP levels in 229E-infected LLC-MK2 cells after 2, 3, and 4 dpi were determined by western blotting. The graph indicates changes in NP/actin ratio (n = 3, normalized with the ratio of G12(S) (−) at 2 dpi as 1) of western blotting experiments. b 229E RNA in the infected cells (left) and in the culture medium (right). c Reduction of 229E virus titers in the presence of G12(S) in the medium. LLC-MK2 cells were infected by 229E for 3 and 4 dpi. d 229E RNA number in the culture media per viral infectious titer are shown. Reduction of SARS-CoV-2 virus RNA (e) and titers (f) in the medium of the virus-infected VeroE6/TMPRSS2 cells (MOI = 0.01) in the presence of 1 and 2 μM G12(S) at 24 and 48 hpi. g SARS-CoV-2 medium RNA per infection titer (pfu). Data are shown box-and-whisker plots including all data plots with the P-values. Statistical analyses of the G12(S) effect by Two-Way ANOVA (a n = 3, F(1,4) = 116.5, P = 0.0004); b left, n = 3, F(1, 2) = 1015, P < 0.0001; b right, n = 3, F(1,4) = 60.59, P = 0.0015; c n = 3, F(1,4) = 13.62, P = 0.0210; d n = 3, F(1, 4) = 818.5, P < 0.0001) or by REML model (e n = 3, F(2, 6) = 6.227, P = 0.0344; f n = 3, F(2, 6) = 11.20, P = 0.0094; g n = 3, F(2, 12) = 24.12, P < 0.0001). Sidak’s multiple comparison test (a–c) and Dunnett’s multiple comparison test (e–g) were used and the P-values are indicated.

Fig. 4. Effect of G12(S) on the intracellular NP distribution, and in vitro NP-vRNA interaction.

a–d Subcellular localization of OC43 NP and EIGIC-53 or ER (KDEL) proteins. A549 cells infected with OC43 (MOI = 1) were cultured for 48 h. The cells were treated with 2 μM G12(S) for the next 24 h of the culture, and fixed. Immunostaining was performed for OC43 NP with KEDL proteins (a and b) or ERGIC53 (c and d). See Supplementary Fig. 5e for the specificities of the antibody used for OC43 NP, ERGIC-53, and ER protein (anti-KDEL antibody). e Experimental overview of LLPS formation and G12(S) treatment on the LLPS. Droplets of SARS-CoV-2 NP were formed by LLPS in the presence of 1 µM FITC-32 base of the 3’-untranslated region of SARS-CoV-2 RNA and 40 µM SARS-CoV-2 NP overnight. f Effect of TAMRA-G12(S) on droplets of SARS-CoV-2 NP formed by LLPS in the presence of 1 µM FITC-leveled vRNA. Fluorescence and differential interference contrast (DIC), TAMRA (magenta) and FITC (Green) and merged (Merge) images are shown. TAMRA-G12(S) (final 1, 3, and 6 µM) were added and observed at 25 °C using fluorescence and DIC microscopy at the indicated time. See Supplementary Fig. 6 for the time-dependent image data (10 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 7 h). g The supernatant (soluble fraction) was collected from the wells forming LLPS 1 h after addition of TAMRA-G12(S) (final 0, 3, and 6 µM), and the adsorbed fraction was collected by suspending the wells in LLPS buffer containing 0.1%SDS. These samples were separated in 7.5 M urea/10 mM KCl containing 15% PAGE. Fluorescent images were taken with a ChemiDoc Touch MP (BioRad) using filter sets for Alexa546 (left) and fluorescein (right). Tamura and FITC fluorescence were observed using the Aelxa546 mode. FITC fluorescence was observed in fluorescein mode (center). An overlay image is shown (right). The positions of ssRNA marker lengths are indicated.

Fig. 5. Strong inhibition of IAV propagation by G12(S).

a, b Effect of IAV protein and RNA levels (NP and HA) in IAV (A/Aichi/2/68 (H3N2))-infected MDCK cells by 2 μM G12(S). The time-dependent levels at 15, 18, 21, and 24 hpi were determined. Relative NP and HA levels were quantified from the western blotting images (normalized by actin). The RNA copy numbers in cells were determined as described in the Materials and Methods (b). c Level of IAV RNA (NP and HA) in the culture medium of each well at 15, 18, 21 and 24 hpi. d To avoid repeated infections, MDCK cells were infected with IAV at an MOI of 1 in medium lacking trypsin, which is required for nascent IAV infectivity. e Effect of 0.5 and 2 μM of G12(S) on (+) strand and (−) strand NP RNA levels in cells at 4 and 6 hpi were determined. f Inhibitory effect of G12(S) on (−) strand NP RNA in the culture medium at 6 hpi was determined. g Pfu of the culture media of MDCK infected IAV (H3N2 Aich) (MOI = 0.01) at 24 hpi was determined in the presence of 2 µM G12(S). h Pfu of the culture media of MDCK infected (MOI = 0.01) with mouse-adapted A/California/04/09 (H1N1pdm09) and mouse-adapted A/Aichi/2/68(H3N2) (24 hpi). i Dose-response (inhibition of CPE) curve obtained by treating IAV (A/Aichi/2/68 (H3N2))-infected MDCK cells (MOI = 0.01, N = 3) with oseltamivir (Tamiflu), laninamivir (Inavir), and G12(S) at 20 μM and six 4-fold dilutions for 27 h are shown. Because IAV infection caused cells to detach from the plate surface, cells remaining on the plate were quantified by crystal violet staining and their absorbance. The dose-response curves (n = 4, +/−SD) were fitted by nonlinear regression (curve fit) using log (drugs) vs. response variable slope (four parameters). The results and EC₅₀ were obtained by three independent experiments (See Supplementary Fig. 9). j Pfu of the culture medium of MDCK infected IAV (H3N2 Aich) (MOI = 0.01) in the presence of 0.5, 1.0 and 2.0 µM G12(S), Tamiful and Inavir at 22 hpi was determined. Data (a–c, e–h) are shown by box-and-whisker plots including all data plots with the P-values. Statistical analyses of the G12(S) effect by Two-Way ANOVA (a left, n = 3, F(3, 12) = 36.68, P < 0.0001; a right, n = 3, F (1, 4) = 38.59, P = 0.0034; b left, n = 3, F(1, 4) = 2025, P < 0.0001; b right, n = 3, F(1, 4) = 712.1, P < 0.0001; c left, n = 3, F(1, 4) = 528.9, P < 0.0001; c right, n = 3, F(1, 4) = 410.5, P < 0.0001; e left, n = 6, F(2,15) = 1.239, P = 0.3177; e right, n = 6, F(2, 15) = 0.2740, P = 0.2740), or by One-Way ANOVA (f n = 6 or 8, F(2, 17) = 21.92, P < 0.0001; h left, n = 3, F(2, 6) = 18.06, P = 0.0029; h right, n = 3, F(2, 6) = 16.68, P = 0.0035). Two-tailed t-test was used for g (F(2, 2) = 2006, P-value = 0.0010). Sidak’s multiple comparison test (a–c), Tukey’s multiple comparison test (e) or Dunnett’s multiple comparison test (f, h) were used and the P-values are indicated.

Fig. 6. Effect of IAV infection on the localization of mEGFP-NP and mCherry-Rab11A.

a Construction of a dicistronic plasmid that expresses mEGFP and mCherry-Rab11A simultaneously. The gene was driven by a CMV promoter and an EMCV IRES was inserted before the second cistron in mCherry-Rab11A. At 1 h after IAV infection (MOI = 1), the plasmid was transfected to the A549 cells. The cells were further cultured for 23 h. The medium was changed to phenol red-free MEM containing glutamine and NucBlue (Hoechst 33342) for staining the nucleus. b Effect of 2 μM G12 on the colocalization of mEGFP-NP (green) and mCherry-Rab11A (red). Non-infected cells (i and iii), IAV-infected cells (ii and iv), and each mCherry, EGFP, and the superimposed images of all including NucBlue (blue) are shown. Multiple images taken by same condition are shown in Supplementary Fig. 10. c, d Effect of 85 min treatment of G12(S) on IAV-induced punctate appearance and movement of mCherry-Rab11A. Superimposed images of mEGFP-NP and mCherry-Rab11A are shown in the left panels. Images at 1-s intervals for mCherry–Rab11A and its movement are shown under the conditions of IAV infection (c) uninfected cells (d) after treatments of 2 μM G12(S) for 15 and 85 min (c) and 40 min (d). The parts of images of the mCherry-Rab11A are 200% magnified. Scale bars indicate 10 and 2.5 μm, respectively. Corresponding movie files (Supplementary Movies 1–4) are indicated in c and d.

Fig. 7. G12(S) forms G-tetrads and may have a left-handed G-quadruplex structure similar to Block2Δ.

a The four guanine form a planar G-tetrad structure through Hoogsteen hydrogen bonds and are stabilized by alkali metal ions (e.g., K⁺ and Na⁺). Guanosine-rich RNA oligomers form several G-tetrads with a parallel orientation to form G-quadruplex structures. The structure of Block2Δ (GTGGTGGTGGTG) consists of two subunits of two-layered parallel-strand G-quadruplex structure with left-handed helicity. b CD spectra of G12(S) in 10 mM Tris/HCl (pH 7.0) (black curve), 30 mM NaCl (blue curve) or 30 mM KCl (yellow curve). G12(S) (4 μM) was incubated overnight in 10 mM Tris/HCl, and then further incubated overnight with 30 mM NaCl or 30 mM KCl. c Nucleotide sequences of G12(S) and BL2(S). The Tm of G12(S) in 30 mM KCl or 30 mM NaCl in 10 mM Tris/HCl (pH7.0) obtained for the first and second temperature increases are shown (see Supplementary Fig. 12). Helicity of G-quadruplexes determined by CD spectroscopy are shown as left-handed parallel (L) and right-handed parallel (R). d CD spectra of BL2(S). BL2(S) was incubated in H₂O overnight (blue curve), and then further incubated in Na-K phosphate buffer (orange curve), 30 mM KCl-10 mM Tris/HCl (pH 7.0; yellow curve) or 30 mM NaCl-10 mM Tris/HCl (pH 7.0) (gray curve) for 1 h. e, f Mobility shift of G12(S) and BL2(S) in native PAGE. G12(S) and BL2(S) at 4 μM (20 pmols) pretreated with 0, 5, 10, 20, or 40 μM of NMM and then with 10 mM KCl-Tris/HCl for 20 min were separated on a 15% PAGE at a constant voltage of 120 mV for 100 min using Tris/borate containing 10 mM KCl (e) or 10 mM NaCl (f). The gels were stained with NMM. The dsRNA marker in the rightmost lane of each gel was pretreated with 200 pmol of NMM prior to PAGE, whereas the second lane from the left was not, which was detected by subsequent DAPI staining (see Supplementary Fig. 11c). g Effects of MNN on CD spectra of G12(S), BL2(S) and ADAM10(S). These PS-RNA were incubated overnight in H₂O, and then incubated with 0, 10 and 20 μM NMM in 10 mM Tris/HCl (pH 7.0) for 30 min and further incubated with 30 mM KCl for 1 h. CD spectra of 0 μM NMM (blue curve), 10 μM NMM (orange curve) and 20 μM NMM (gray curve) are shown. h Possible structural changes induced by NMM and NaCl in the G-quadruplex dimer structure of BL2(S), which may be a structural model for G12(S). NMM and/or NaCl convert the left-handed parallel dimer of BL2(S) formed in the presence of K⁺ into a right-handed parallel monomer.

Fig. 8. Correlation between anti-IAV activities of 12-18mer PS-RNA and intracellular G-quadruplex levels.

a Nucleotide sequences of left-handed parallel DNA Block2, Block2Δ and corresponding BL2-RNA and PS-modified BL2 (BL2(S)). b Anti-IAV activity was examined by the Block2 and Block2Δ DNAs (2 μM). c Nucleotide sequences of PS-RNA version of known sequences (12mer to 18mer). Gq(S) is a 12-nt sequence consisting of UUU flanked by two GGGs, with U and UU at the 5’ and 3’ ends, respectively. MTA2 and ADAM10 are found in the 5’-untranslated region of human cellular mRNAs that can form G-quadruplex structures. 93del is derived from an aptamer DNA isolated as HIV-1 integrase inhibitor and inhibits HIV-1 replication. 93del forms a parallel-fold G-quadruplex structure. G15 oligo DNA has been found to be the shortest poly-G sequence that form a strict G-quadruplex structure. All PS-RNA showed right-hand parallel CD spectra (R) (see Supplementary Fig. 13b). d Anti-IAV activities of PS-oligonucleotide (2 μM) examined. e, f A549 cells were treated with 2 μM PS-RNAs (indicated in the figures) and stained with QUMA-1. We measured G-quadruplex levels in cells (7-μm thick) by overlaying eight slices acquired with a super-resolution confocal laser scanning microscope (LSM900 with Airyscan 2) using the G-quadruplex-specific fluorescent dye QUMA-1. Shown are separate images (e) overlay images (f) of eight planes (z-direction) of QUMA-1 (yellow) and DAPI staining (blue). AF610 (Excitation = 612 nm, Emission = 630 nm, Detection = 590–700 nm) used for QUMA-1 observation. Black and white of AF610 set at approximately 4000 and 12,000 (f). g PS-RNA-dependent QUMA-1 fluorescence by adjusting the brightness threshold to 4000 to exclude nucleus autofluorescence and fluorescence of cells untreated with PS-RNA (see Supplementary Fig. 13d, original data were available at 10.5061/dryad.8931zcs1s). The total QUMA-1 brightness value for each overlay image was divided by the number of nuclei to obtain the QUMA-1 brightness value per cell. To average out variability due to cell size, the QUMA-1 brightness values were divided by the average size of nuclei in each overlay image. Independent data sets (2~6 overlay images containing 36–99 cells) are shown by box-and-whisker plots including all data plots. Statistical analyses of the G12(S) effect by One-Way Repeated measures ANOVA (b F(2, 6) = 2.263, P-value = 0.1852; d F(7, 19) = 551.1, P < 0.0001; g F(7, 20) = 6.133, P = 0.0006), and Tukey’s multiple comparison test was used. “ns” indicates P-value > 0.5 (b). P-values of d and g are indicated in Supplementary Figs. 13c and 13e, respectively. The letters shown in d and g are classifications created using Compact Letter Display (CLD) in GraphPad Prism 10, which is another way of grouping based on significant differences, e.g., between A and B, the P-value is less than 0.05, but between A and AB or AB and B, the P-value is greater than 0.05. h Correlation between IAV antiviral effect and cytoplasmic G-quadruplex level was examined using Pearson’s correlation coefficient. The average IAV reduction value and QUMA-1 fluorescence intensity are plotted, and the color of each point indicates the PS-RNA treatment corresponding to the color used in graphs d and g.

Fig. 9. G-quadruplex formation is required for anti-IAV activity.

a Nucleotide sequences of PS-RNAs examined. BL2-14(S) and 5U-BL2-14(S) correspond to Block 2 and 5’T-Block 2 DNA oligomer, respectively. BL2-UU25(S) is the 5’-teminal half of 2xBlock 2 T2,5,16,19TT DNA. b CD spectra of BL2-14(S), BL2-UU25(S) and 5U-BL2(S) in H₂O or 10 mM Tris/HCl (pH 7.0) containing 30 mM KCl. c Anti-IAV activity was examined by the PS-RNAs (2 μM). d, e QUMA-1-stained cells were observed as described legend to Fig. 8. Independent data sets (3 overlay images containing 30–65 cells) were shown by box-and-whisker plots including all data plots with the P-values. Statistical analyses of the G12(S) effect by One-Way Repeated measures ANOVA (c F(3, 26) = 63.85, P < 0.0001; e F(3, 13) = 14.38, P = 0.0002) and Tukey’s multiple comparison test was used. P-values of c and e are indicated in Supplementary Figs. 14a and b, respectively. The letters shown in (c and e) are classifications created using CLD (see legend to Fig. 8).

Fig. 10. Docking models SARS-CoV-2 N protein dimer in complex with G-quadruplex 12-mer RNA, and Schematic diagrams of the possible antiviral effects.

a Anti-coronavirus (OC43) activity of BL2(S). MRC-5 cells were infected with OC43 (MOI = 0.01) and the culture medium was collected at 4 pdi. Pfu was determined by plaque assay using MRC-5 cells. b Docking models of the SARS-CoV-2 N protein dimer in complex with BL2-RNA molecules. Docking was performed using HADDOCK server and the three docking poses were superimposed. The proteins are depicted in gray with ribbon model, and the unstructured regions were omitted for clarity. The bound BL2-RNAs are shown in orange. c The replication and transcription of CoV vRNAs occur in DMVs derived from the ER membrane. Viral vRNA are transported to the cytoplasm, where they perform NP binding and form RNP complexes. This step may be inhibited by G12(S) and the resulting putative immature RNPs may be then transported to the ER-Golgi intermediate compartment (ERGIC). Virus particles are formed within the ERGIC and secreted as noninfectious coronaviruses (RNAs) outside the cell. d Replication cycle for IAV. Upon the entry and shedding of IAVs, eight vRNPs are transported into the nucleus, where mRNA and complementary RNA (cRNA) are synthesized. The newly synthesized NPs and IAV RNA polymerase subunits (PA, PB1, and PB2) are imported into the nucleus for vRNA replication and vRNP formation. The assembled vRNPs are exported to the cytoplasm and attached to the recycling endosome-specific G-protein Rab11A (red circle). The Rab11A-vRNPs are transported to the plasma membrane, where the viral is packaged and secreted outside the cells. G12(S) may inhibit vRNA-NP interaction, which is required for Rab11-dependent transport.