Targeting genomic SARS-CoV-2 RNA with siRNAs allows efficient inhibition of viral replication and spread

Abstract

A promising approach to tackle the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) could be small interfering (si)RNAs. So far it is unclear, which viral replication steps can be efficiently inhibited with siRNAs. Here, we report that siRNAs can target genomic RNA (gRNA) of SARS-CoV-2 after cell entry, and thereby terminate replication before start of transcription and prevent virus-induced cell death. Coronaviruses replicate via negative sense RNA intermediates using a unique discontinuous transcription process. As a result, each viral RNA contains identical sequences at the 5' and 3' end. Surprisingly, siRNAs were not active against intermediate negative sense transcripts. Targeting common sequences shared by all viral transcripts allowed simultaneous suppression of gRNA and subgenomic (sg)RNAs by a single siRNA. The most effective suppression of viral replication and spread, however, was achieved by siRNAs that targeted open reading frame 1 (ORF1) which only exists in gRNA. In contrast, siRNAs that targeted the common regions of transcripts were outcompeted by the highly abundant sgRNAs leading to an impaired antiviral efficacy. Verifying the translational relevance of these findings, we show that a chemically modified siRNA that targets a highly conserved region of ORF1, inhibited SARS-CoV-2 replication ex vivo in explants of the human lung. Our work encourages the development of siRNA-based therapies for COVID-19 and suggests that early therapy start, or prophylactic application, together with specifically targeting gRNA, might be key for high antiviral efficacy.

Graphical Abstract

Exclusive targeting of SARS-CoV-2 genomic RNA enables most efficient suppression of viral replication and virus-induced cell death.

Figure 1.

Effect of targeting genomic SARS-CoV-2 RNA with siRNAs on viral replication and cytopathy. (A, top) Experimental setup used in (B–D). VeroE6 cells were transfected with siRNAs targeting ORF1 (siORF1) 16h before infection with recombinant, GFP-expressing SARS-CoV-2 (rSARS-CoV-2-GFP; MOI 1) and number of GFP⁺ positive cells quantified. Cells receiving no treatment (untreated), transfection reagent only (Mock) or a control siRNA (siCtrl) served as controls. (A, bottom) Schematic representation of gRNA, as well as sgRNAs. Note that ORF1 (blue) is only part of full-length gRNA but not sgRNAs. GFP, green fluorescent protein. (B) Kinetic of viral spread showing number of GFP⁺ cells determined by automated quantification using the integrated Incucyte S3 software. (C, D) GFP expression 24h after infection with rSARS-CoV-2-GFP. (C) Exemplary fluorescence microscopy pictures. Bar at lower right indicates 0.1 mm length and (D) quantification of GFP⁺ cells. (E) Same experimental setup as in (B–D) but cells were infected with wildtype SARS-CoV-2 (MOI 0.1) and lysed after 24 h to quantify genomic SARS-CoV-2 RNA from cell lysate by RT-qPCR. (F, G) siRNAs used in (B–E) were pooled and transfected into VeroE6 cells 6h before infection with wildtype SARS-CoV-2. Cells were lysed at different time points after infection and SARS-CoV-2 (F) gRNA as well as (G) sgRNAs quantified by RT-qPCR. (H, I) VeroE6 cells were transfected with siRNAs 6h before infection with wildtype SARS-CoV-2 (MOI 1) and dead cells visualized using the Incucyte® Cytotox Red Dye and quantified using the Incucyte S3. (H) Exemplary fluorescent microscopy pictures taken at 56h p.i. Dead cells are shown in red. Bar at lower right indicates 100 μm length. (I) Time kinetic of dead cells quantified every 4h over a period of 3 days. (B, G, I) Mean of triplicates for each treatment group is shown, error bars indicate SEM. Bars in (D–F) show median. Statistical differences were calculated using (B, G, I) repeated measures one-way Anova or (D–F) regular one-way Anova with Dunnett′s multiple comparison correction. M, Mock; –, untreated; O1-3, ORF1-specific siRNAs 1–3; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2.

Evaluation of SARS-CoV-2 negative sense RNA as siRNA target. (A, B) Kinetics of negative and positive sense SARS-CoV-2 RNAs following wildtype SARS-CoV-2 infection (MOI 0.1) of VeroE6 cells. Negative and positive sense RNAs were individually transcribed to cDNA by using either poly A or poly T primers and (A) gRNA and (B) sgRNAs quantified by RT-qPCR. (C) Experimental setup to determine siRNA strand specific activities. Luciferase reporters with incorporated positive or negative sense N sequences in the 3′UTR of Renilla luciferase were co-transfected with siRNAs into HEK293T cells and (D) luciferase activity measured after 48 h (E) siRNAs were transfected into VeroE6 cells 6 h before infection with wildtype SARS-CoV-2 (MOI 0.1) and 24 h p.i. sgRNAs quantified from cell lysate using RT-qPCR. (F) Same setup as in (E) but VeroE6 cells were infected with rSARS-CoV-2-GFP (MOI 1.0) and GFP⁺ cells quantified every 4 h. All experiments were performed with three biological replicates. Graphs in (A, B, D, E) show mean and error bars SEM. Statistical differences were calculated using (E) Regular or (F) repeated measures one-way Anova with Dunnett′s multiple comparison correction. Co-transf., co-transfection; M = mock-transfected; n.s., non-significant, *P < 0.05; ****P < 0.0001

Figure 3.

Targeting common regions of SARS-CoV-2 transcripts allows simultaneous suppression of gRNA and sgRNAs, but leads to impaired antiviral activity. (A) Schematic presentation of SARS-CoV-2 transcripts with sequences that are found in several transcripts shown in orange or red, and sequences that are exclusively part of viral gRNA shown in blue. (B) Effect of siRNAs targeting ORF1 which is only part of full-length SARS-CoV-2 gRNA or targeting sequences common within gRNA and sgRNAs. VeroE6 cells were infected with wildtype SARS-CoV-2 (MOI 0.1) and 3 h p.i. transfected with siRNA pools (containing three siRNAs each) specific for indicated genomic regions of SARS-CoV-2. At 24 h p.i., viral gRNA and sgRNAs were quantified by RT-qPCR. gRNA levels are shown relative to 18S rRNA and sgRNA relative to gRNA. (C) VeroE6 cells were infected with rSARS-CoV-2-GFP (MOI 1) and 3 h later transfected with individual siRNAs targeting indicated genomic regions of SARS-CoV-2. GFP⁺ cells were quantified every 4h (for full data see Supplementary Figure S3C) and virus spread quantified by fitting an exponential curve and calculating the doubling time. Dots represent median of three biological replicates each. Name of siRNA is given by red and blue labeling; L1–3; Leader-sequence specific siRNAs 1–3; N1–3, N-specific siRNAs 1–3; U, 3′UTR-specific siRNAs 1–3; O1–3, ORF1-specific siRNAs 1–3. (D) Comparison of siRNA efficacy against luciferase reporters or SARS-CoV-2 infection. To determine activity against luciferase reporters, each siRNA was transfected together with the respective luciferase reporter into HEK293T cells and luciferase activity measure after 48 h. To measure antiviral activity, experimental setup as described under (C) was used, and GFP⁺ cells quantified at final time point (68 h). Each dot represents median of three biological replicates. (E) VeroE6 cells were transfected with siRNA pools and infected with wildtype SARS-CoV-2 (MOI 0.1) after 6 h. Viral gRNA was quantified relative to 18srRNA at given time points using RT-qPCR (F, G). Effect of siRNA treatment on SARS-CoV-2 induced cytolysis. VeroE6 cells were transfected with siRNA pools and infected with wildtype SARS-CoV-2 (MOI 1) after 6 h. Virus-induced cell death was analysed using the Incucyte^® Cytotox Red Dye at 56 h p.i. (F) Exemplary fluorescence microscope images showing dead cells in red. Bars in lower right of images represent 100 μm. (G) Number of dead cells were quantified using the Incucyte S3 analyzing system. Bar in (C, D) shows median. (B, E, G) show mean ± SEM. Statistical differences were calculated using (B, G) one-way Anova, or (E) repeated measures Anova with Dunette′s multiple comparison correction and in (C, D) using Student's t-test for independent samples. All experiments were performed using three biological replicates. n.s., non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4.

Subgenomic RNAs out-compete and impair antiviral activity of siRNAs. (A, B) VeroE6 cells were transfected with siRNAs targeting sgRNA and gRNA (N2) or exclusively gRNA (O2), infected 6h later with rSARS-CoV-2-GFP and the number of GFP⁺ cells was determined 24 h p.i. (A) siRNAs were transfected at a concentration of 1 nM, and cells were infected with MOIs of 0.03, 0.3 and 3. (B) siRNAs were transfected at varying concentrations ranging from 0.1 to 100 nM and VeroE6 were infected with a MOI of 0.3. (C) Comparison of mean inhibitory concentrations (IC₅₀) of siRNAs determined using luciferase reporters (left graph) or rSARS-CoV-2-GFP (right graph). Full data is shown in Supplementary Figures S4 and S5. For experimental details see Materials and Methods section. (D, E) Competition experiment to determine effect of SARS-CoV-2 replication on RNAi silencing efficacy. (D) HEK293T cells were co-transfected with siRNAs against different target region as well as luciferase reporters with incorporated binding sites for the co-transfected siRNA. After 6h, cells were infected with wildtype SARS-CoV-2 (MOI 0.1) and (E) luciferase activity determined from cell lysate 24 h p.i.. Statistical differences were calculated using Student's t-test for independent samples; n.s., non-significant, *P < 0.05; **P < 0.01.

Figure 5.

Chemically modified siRNA inhibits SARS-CoV-2 replication ex vivo in the human lung. (A) ORF1-targeting siRNAs were chemically modified using a clinically validated chemistry (for details, see Material and Methods and Supplementary Table S4) and the activity compared to chemically non-modified versions of the siRNAs using luciferase reporters. For this, siRNAs and luciferase reporter plasmids were co-transfected into HEK293T cells and after 24 h luciferase activities determined. Values were normalized to a control group transfected with the respective luciferase reporter and the control siRNA with identical chemistry. (B) Effect of chemical modifications on the duration of RNAi-silencing by siRNA O3 was compared using the same experimental setup as in (A), and luciferase activity was determined at indicated time points. (C) Antiviral activity of the modified and non-modified version of siRNA O3 were compared using the rSARS-CoV-2-GFP model. siRNAs were transfected into VeroE6 at a concentration of 50nM. 6h later, cells were infected with rSARS-CoV-2-GFP (MOI1), and GFP⁺ cells were quantified using the Incucyte S3 system. (D) To validate the approach in a highly relevant model of the human lung, the chemically modified siRNA O3 was complexed with polyethylenimine (PEI), and transfected into human precision cut lung slices (hPCLS; 100nM), which were infected with wildtype SARS-CoV-2 (MOI 1) 6h later. RNA was extracted from hPCLS harvested 24h p.i. and viral replication quantified by RT-qPCR for SARS-CoV-2 gRNA (normalized to β-actin expression). Experiments shown in (A–C) were performed using three biological replicates, (D) using five replicates. Horizontal bars in (A, D) indicate mean, error bars in (B–D) S.E.M. n.s., non-significant; **P < 0.01; ***P < 0.001.