Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome

Abstract

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.

Keywords: LNA; SARS-CoV-2; astrovirus; coronavirus; gapmer; plus-strand RNA virus; s2m; therapeutic oligonucleotides.

FIG 1

s2m is a conserved structural element in the SARS-CoV-2 genome. (A) Sequence alignment of the s2m element in the 3′ UTRs of SARS-CoV-2 and SARS-CoV. Lines indicate base-pairing regions within the element. (B) Crystal structure of the SARS-CoV s2m element (adapted from PLoS Biology [13]). (C) SHAPE chemical probing of the 3′ UTR of SARS-CoV-2. RNA was denatured and refolded in the presence of 100 mM K⁺ and 0.5 mM Mg²⁺ and then incubated with NAI (+NAI channel) or DMSO control (−NAI channel). NAI modification was detected by reverse transcription stalling and gel-based analysis. Sequencing lanes were generated by adding ddT (for A), ddG (for C), ddC (for G), and ddA (for U) when reverse transcription was performed. (D) Annotation of SHAPE signal on the s2m structure. (E) Bar plot showing the reactivities of structural profiling of gel-based analysis in our study (C) and in the SHAPE-MaP experiment described by Manfredonia et al. (23). The s2m structure is highlighted by shading in green. (F) Representative cryoEM image of the SARS-CoV-2 3′ UTR (220 nt) at a 2.5-μm defocus. The red arrows indicate features that likely correspond to views along the long axis of duplex regions. Bar, 50 nm. (G) 2D class averages and (H) 3D reconstructions were calculated by cryoSPARC 2.15.0.

FIG 2

Antisense oligomers direct RNase H cleavage of the s2m element and a conserved single-stranded region (ss3) in vitro. (Top) Design of the six gapmers complementary to the s2m, as well as a nonspecific control gapmer “scr” used in this study (Table 3). The LNA is indicated in orange, s2m-specific phosphorothioate-linked DNA in pink/purple, other phosphorothioate-linked DNA in gray (“scr” and “all”), and phosphodiester-linked DNA in light gray (“some”). RNase H cleavage of the isolated s2m (A and D), 3′ UTR (B and E), the extended 3′ UTR (F), and the predicted single-stranded region ss3 (C). Three target-to-gapmer molar ratios were tested: 1:0.5, 1:1, and 1:2. Incubation of RNA target with RNase H alone does not lead to cleavage (RNase H, last lane) and is not driven by control gapmers with scrambled DNA sequence (“scr”). Incubation of RNA target with gapmer without the addition of RNase H does not lead to degradation either, but it does lead to the appearance of a retarded band that likely corresponds to the target-gapmer duplex.

FIG 3

SHAPE probing reveals RNA structure changes induced by LNA gapmers. (A, C, E, and G) SHAPE probing of SARS-CoV-2 3′ UTR structure in the presence or absence of the gapmer indicated. RNA was denatured and refolded in the presence of 100 mM K⁺ and 0.5 mM Mg²⁺, then incubated with different gapmer-to-target molar ratios (0, 0.5, 1, and 2), and probed using NAI. (B, D, F, and H) Quantification of results in panels A, C, E, and G, respectively. Analysis of the differences in SHAPE signal from SARS-CoV-2 3′ UTR alone and in the presence of 2× molar excess of the gapmer. (A and B) The presence of gapmer 1 induced an increase in SHAPE signal at positions 70 to 74 and 79 to 94 (highlighted in orange), indicating that these nucleotides are more unstructured. A strong decrease in SHAPE signal was observed at positions 60 to 69 (highlighted in blue), indicating decreased accessibility of these bases, which could be caused by their base-pairing with the gapmer. (C and D) The reactivity profile in D is similar to that in B, due to the similar target regions of gapmer 1 and gapmer 2. (E and F) In the presence of gapmer 3, nucleotides at positions 69 to 75 are more structured, while nucleotides at positions 76 to 94 are less structured, as indicated. (G and H) No significant differences in SHAPE signal could be detected in the presence or absence of the nonspecific control gapmer “all,” indicating that it is unable to cause structural changes in the SARS-CoV-2 3′ UTR.

FIG 4

Gapmer-induced reduction of protein levels in cell reporter assays. (A) Schematic of the GFP reporter assay. (B) Gating and data visualization strategy of the reporter assay data. The main cell population was identified and gated on forward and side scatter using the Auto Gate tool and plotted as a histogram to visualize GFP intensity of cells. (C) HeLa and A549 cells containing a genomic insertion of a GFP reporter construct without additional insertion in its 3′ UTR (GFP), with the s2m sequence in its 3′ UTR (GFP-s2m), or with a scrambled sequence insertion in its 3′ UTR (GFP-s2m-scr) were analyzed by flow cytometry. Data are representative of two independent experiments. (D to G) Flow cytometry analysis of the GFP-expressing cells. HeLa (D) and A549 (F) cells containing a genomic insertion of a GFP reporter construct with the s2m sequence in its 3′ UTR (GFP-s2m) were transfected with a 20 nM concentration of the indicated gapmers and analyzed 72 h posttransfection by flow cytometry. Treatment with gapmers against the s2m element, but not a nonspecific control gapmer, induced reduction in fluorescence. HeLa (E) and A549 (G) cell lines containing a control genomic insertion of a GFP reporter construct with a scrambled sequence inserted in its 3′ UTR (GFP-s2m-scr) were transfected with 20 nM the indicated gapmers and analyzed 72 h posttransfection by flow cytometry. The control cell lines containing GFP reporter with a scrambled sequence insertion in the 3′ UTR show no appreciable change in fluorescence upon treatment with the gapmers targeted against the s2m element. Data are representative of three independent experiments (D to G).

FIG 5

Inhibition of astrovirus replicon activity by gapmers targeting the SARS-CoV-2 s2m RNA element. (A) Schematic of the SARS-CoV-2 and human astrovirus 1 (HAstV1) genome organization. The lowest panel represents the astrovirus replicon (pO2RL). FS, frameshift signal; SG, subgenomic; RLuc, Renilla luciferase; RdRp, RNA-dependent RNA polymerase. The presented virus and replicon genomes are not to scale. (B) Conservation of the s2m 3′ UTR element (two-dimensional representation) between HAstV1, SARS-CoV, and SARS-CoV-2. In the astrovirus replicon, the HAstV1 s2m was switched for the SARS-CoV or SARS-CoV-2 s2m; the wild-type and chimeric replicons are indicated below. Gapmers 1, 2, and 3 and gapmers 4, 5, and 6 are color coded in light, medium, and dark magenta, respectively. (C) Luciferase activity of the wild-type, chimeric, and replication-deficient (RdRp GDD motif mutated to GNN) astrovirus replicons measured in Huh7.5.1 (dark blue bars) and HEK293T (light blue bars) cells. (D) Inhibition of the astrovirus chimeric replicon containing the SARS-CoV-2 s2m by gapmers at a 0.5 to 500 nM concentration range. (E) Inhibition of the astrovirus chimeric replicon containing SARS-CoV s2m by gapmers at a 0.5 to 500 nM concentration range. For panels D to E, data are means ± standard deviations (SD) from 3 biologically independent experiments; full data and statistical analyses are provided in Tables 4 and 5. Replicon activity is presented as the ratio of Renilla (subgenomic reporter) to firefly (cap-dependent translation, loading control) luciferase activity, normalized by the same ratio for the untreated control replicon.

FIG 6

Testing the cytotoxic and off-target effects of gapmers. (A) Toxicity assay for gapmer-treated cells. Cells were treated with 0.5 to 500 nM gapmers for 24 h. Supernatant was used to measure cell viability, calculated as the ratio of released to total lactate dehydrogenase (LDH) activity; “max” refers to the maximum LDH measured for fully lysed cells. (B) Effect on translation measured as a readout of capped T7 RNA encoding firefly luciferase at a 0.5 to 500 nM gapmer concentration, normalized to the value for untreated cells. All data are means ± SD from 3 biologically independent experiments.

FIG 7

Inhibition of SARS-CoV-2 growth by gapmers targeting the s2m RNA element. (A) Graphic representation of the high-content screening assay experiment workflow: transfection of Vero E6 cells with gapmers followed by infection with SARS-CoV-2, fixation of the plate, labeling, and screening. A representative image from the immunofluorescence-based detection of SARS-CoV-2 infection of Vero E6 cells is shown at the bottom. (B) Vero E6 cells were transfected with gapmers 1 to 6 against the s2m element or control gapmers “all” and “scr” at 0.25 μM, 0.5 μM, and 1 μM final concentrations, infected with SARS-CoV-2, fixed, labeled, and analyzed. Results are presented as means ± SD for 3 biological replicates; signal was normalized to a no-gapmer control. Cell viability was evaluated using the DRAQ7 signal normalized to that in mock-treated wells. P values are from two-tailed t tests with separate variances (ns, not significant [P > 0.05]; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (C) Toxicity assay for gapmer-treated Vero E6 cells in the absence of transfection reagent. Cells were treated with 0.25 μM, 0.5 μM, and 1 μM gapmers for 24 h. Supernatant was used to measure cell viability, calculated as the ratio of released to total lactate dehydrogenase (LDH) activity. “max” refers to maximum LDH measured for fully lysed cells.