Cells and viruses
HEK293T (CRL-1573), Vero E6 (CRL-1586) and MDCK-NLB-2 (CCL-34) cells were obtained from American Type Culture Collection (ATCC) and were maintained according to ATCC instructions. A549-Dual (a549d-nfis) and THP1-Dual (thpd-nfis) were obtained from InvivoGen and maintained according to the manufacturer’s instructions. All cell lines used in the present study were routinely checked for Mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza) and were authenticated by the respective vendors. All cell lines used for SARS-CoV-2 experimentation, including Huh-7, ACE2-TMPRSS2-Huh-7.5 and ACE2-A549 cells, were maintained at 37 °C in complete Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Invitrogen), penicillin and streptomycin (Gibco) and Hepes buffer (Gibco). The ACE2-A549 cells were specially engineered to overexpress the ACE2 receptor in human alveolar basal epithelial cells (A549), whereas the ACE2-TMPRSS2-Huh-7.5 cells were a kind gift of the Catherine Blish laboratory and engineered to overexpress both hACE2 and human TMPRSS2 receptors. A549-Dual and THP1-Dual cells were purchased from InvivoGen.
WT influenza A/PR/8/34 (PR8) H1N1 virus (ATCC-VR-95) and the tissue-culture-adapted PR8 virus (ATCC-VR-1469) were purchased from ATCC. PR8 mutant viruses were generated using an eight-plasmid reverse genetic system as previously described38. Tissue-cultured adapted influenza A/Hong Kong/8/68 (HK68) H3N2 virus (ATCC-VR-1679), A/Virginia/ATCC6/2012 (H3N2) virus (ATCC-VR-1811), A/Virginia/ATCC1/2009TC (H1N1) virus (ATCC-VR-1736) and A/Wisconsin/33 (H1N1) virus (VR-1520) were purchased from ATCC. A/California/4/2009 (pH1N1) virus was kindly gifted by E. Govorkova from St. Jude Children’s Research Hospital (Memphis, USA). Viruses were grown and amplified in 10-d-old, specific, pathogen-free, research-grade chicken embryos at 35 °C (Charles River Laboratories; SPAEAS).
WT recombinant SARS-CoV-2 was prepared and handled as described39. The recombinant SARS-CoV-2-Nluc virus is an authentic, fully replicating virus in which ORF7a has been deleted and replaced with Nluc40. The SARS-CoV-2 clinical variant was isolated from a 54-year-old man with melanoma and lymphoma, who received a confirmed COVID-19 diagnosis by PCR in March 2020. Virus sequencing was performed on virus isolated from a nasopharyngeal swab sample in virus transport medium taken after the patient had been infected for 7 months.
Plasmid constructs and cloning
Plasmids were used containing the WT PB2 segments from influenza viruses A/Puerto Rico/8/34 (H1N1) (PR8), A/New York/470/2004 (H3N2) (NY470), A/New York/312/2001 (H1N1) (NY312), A/Brevig Mission/1/1918 (H1N1) (1918), A/California/04/2009 (H1N1) (CA09) and A/Vietnam/03/2004 (H5N1) (VN1203). For the generation of PR8 packaging mutant vRNA, we utilized a Stratagene QuickChange XL site-directed mutagenesis kit for mutagenesis of a pDZ plasmid containing the PB2 gene of PR8 (ref. 38). Sequences of each mutated construct were confirmed by automated sequencing. The eight-plasmid pBD rescue system for A/WSN/33 (H1N1) was kindly donated by A. Mehle. The H275Y NA mutant was generated by QuickChange mutagenesis from the bidirectional pBD plasmids, as described above.
Reverse genetics and virus titrations
Recombinant A/Puerto Rico/8/34 (PR8) virus and recombinant A/WSN/33 (WSN) virus were generated using eight-plasmid reverse genetic systems38. Briefly, 1 × 106 cells of a HEK293T/MDCK co-culture were Lipofectamine 3000 (Invitrogen) transfected with 1 μg of one of each of the eight segments contained within plasmids that utilize a bidirectional dual Pol I/II promoter system for the simultaneous synthesis of genomic vRNA and messenger RNA. For rescue of compensatory PB2 mutant viruses where a nonsynonymous change was required, a WT PB2 protein expression plasmid (Pol II) was co-transfected during virus rescue. Supernatants were collected 24 h post-transfection. PR8 rescue viruses were then inoculated into the allantoic cavities of 10-d-old chicken embryos. WSN rescue viruses were passaged subsequent times on MDCK cells. Rescue of recombinant viruses was assessed by hemagglutination (HA) activity. Each newly rescued virus was further plaque titered and mutations were confirmed by sequencing of mutated genes. Plaque assays were carried out on confluent MDCK cells as described previously41. HA assays were carried out in 96-well round-bottomed plates at room temperature, using 50 μl of virus dilution and 50 μl of a 0.5% suspension of turkey red blood cells (LAMPIRE Biological Laboratories) in PBS.
Isolation of packaged vRNAs
To analyze packaged vRNA for PR8 mutated viruses, 10-d-old eggs were inoculated with approximately 1,000 p.f.u. of recombinant virus and incubated for 72 h. Allantoic fluid was harvested and supernatant was dual clarified by low-speed centrifugation. Clarified supernatant was then layered on a 30% sucrose cushion and ultracentrifuged at 30,000 r.p.m. for 2.5 h (Beckman Rotor SW41). Pelleted virus was resuspended in PBS and TRIzol (Invitrogen) extracted. Precipitated vRNA was resuspended in a final volume of 20 μl of 10 mM Tris-HCl, pH 8.0 and stored at −80 °C. Virus supernatant from LNA-treated cells was harvested 48 h post-infection and subjected to low-speed centrifugation at 1,000 r.p.m., then 10,000 r.p.m. Isolation continued as indicated above.
Quantitative PCR analysis of packaged vRNAs
Approximately 200 ng of extracted vRNA was reverse transcribed using a universal 3′-primer (5′-AGGGCTCTTCGGCCAGCRAAAGCAGG) and Superscript III reverse transcriptase (Invitrogen). The reverse transcription (RT) product was diluted approximately 10,000-fold and used as a template for quantitative (q)PCR. Separate PCRs were then carried out as previously described42 with segment-specific primers. The 10-μl reaction mixture contained 1 μl of diluted RT product, a 0.5 μM primer concentration and SYBR Select Master Mix (Applied Biosystems) which included SYBR GreenER dye, 200 μM deoxynucleoside triphosphates, heat-labile UDG (uracil-N-glycosylase), optimized SYBR Green Select Buffer and AmpliTaq DNA polymerase UP enzyme. Relative vRNA concentrations were determined by analysis of cycle threshold values, total vRNA amount within a sample was normalized to the level of HA vRNA and then percentages of incorporation were calculated relative to the levels of WT vRNA packaging. Viral packaging results represent the averaged levels of vRNA incorporation ± s.d. derived from two independent virus purifications, with vRNA levels quantified in triplicate.
Denaturing RNA gel
Extracted viral RNA (100–300 ng) was diluted with equal volume of NOVEX TBE–Urea sample buffer and incubated at 70 °C for 10 min before separation on a 6% TBE–Urea gel for 18 h at a constant voltage of 80 V. RNA from each sample was run in several dilutions to enable clear visualization of the genomic RNA without over-saturation of the band’s signal. The RNA was visualized by staining for 30 min in 0.5× Tris/borate/EDTA (TBE) buffer supplemented with 0.5 mg ml−1 of ethidium bromide followed by visualization in a GelDoc EZ Imager system (BioRad). Silver staining was performed using the SilverXpress silver stain kit (Invitrogen) according to the manufacturer’s instruction. Silver staining and ethidium bromide staining were compared and shown to have the same linear range of detection (data not shown); ethidium bromide was selected for lane visualization due to a higher background signal in silver staining. The band intensity in each lane was determined using Image Lab Software (BioRad) and an analysis of the intensity of each genomic band relative to the total intensity of all genome segments was determined and normalized to the intensity of the HA band relative intensity.
Strand-specific RT–qPCR
MDCK cells transfected with 1 M LNA9 or Scr. LNA were infected with PR8 virus at an MOI of 0.1 24 h post-transfection. Then 8 h post-infection total cellular RNA was extracted in TRIzol reagent (Invitrogen) and the RNA was purified using the Direct-Zol RNA mini-prep (Zymo Research) according to the manufacturer’s protocol. RT and qPCR were performed according to the literature43. Complementary DNAs of the influenza vRNA and complementary viral RNA (cRNA) were synthesized with tagged primers to add an 18- to 20-nt tag that was unrelated to the influenza virus at the 5′-end (cRNAtag: 5′-GCT AGC TTC AGC TAG GCA TC-3′; vRNAtag: 5′-GGC CGT CAT GGT GGC GAA T-3′). Hot-start RT with the tagged primer was performed as described in Kawakami et al.43 using saturated trehalose. A 5.5-μl mixture containing 200 ng of total RNA sample and 10 pmol of tagged primer was heated for 10 min at 65 °C, chilled immediately on ice for 5 min and then heated again at 60 °C. After 5 min, 14.5 μl of preheated reaction mixture (4 μl of First Strand buffer (5×, Invitrogen), 1 μl of 0.1 M dithiothreitol, 1 μl of dNTP mix (10 mM each), 1 μl of Superscript III reverse transcriptase (200 U μl−1, Invitrogen), 1 μl of RNasin Plus RNase inhibitor (40 U μl−1, Promega) and 6.5 μl of saturated trehalose) was added and incubated for 1 h. RT–qPCR was performed with PowerUp SYBR Green SuperMix (Applied Biosystems) on a BioRad CFX96 Real-Time System. Then 7 µl of a tenfold dilution of the cDNA was added to the qPCR reaction mixture (10 μl of SYBR Green SuperMix (2×), 1.5 μl of forward primer (10 μM) and 1.5 μl of reverse primer (10 μM)). The cycle conditions of qPCR were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The qPCR primers were: PR8 segment 1 (PB2) cRNA: forward: 5′-TCC ACC AAA GCA AAG TAG AAT GC-3′; reverse: 5′-GCT AGC TTC AGC TAG GCA TCA GTA GAA ACA AGG TCG TTT TTA AAC-3′; PR8 segment 1 (PB2) vRNA: forward: 5′-GGC CGT CAT GGT GGC GAA TAG ACG AAC AGT CGA TTG CCG AAG C-3′, reverse: 5′-AGT ACT CAT CTA CAC CCA TTT TGC-3′; PR8 segment 4 (HA) cRNA: forward: 5′-CTG TAT GAG AAA GTA AAA AGC C-3′, reverse: 5′-GCT AGC TTC AGC TAG GCA TCA GTA GAA ACA AGG GTG TTT TTC-3′; and PR8 segment 4 (HA) vRNA: forward: 5′-GGC CGT CAT GGT GGC GAA TAG GAT GAA CTA TTA CTG GAC CTT GC-3′, reverse: 5′-TCC TGT AAC CAT CCT CAA TTT GGC-3′.
Animals
All animal studies were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Stanford University Administrative Panel on Laboratory Animal Care and by the Utah State University Institutional Animal Care and Use Committee. Animals were housed in disposable cages connected to an Innorack IVC-ventilated rodent housing system under 12 h light:dark cycle at 24 °C and 25–30% humidity. Healthy age-matched female BALB/c mice aged 6–8 weeks (Jackson Laboratories) were randomly separated into groups for infection/treatment or used as uninfected/nontreated controls. Treatment groups were not blinded to the investigators. Mice were identified with tag numbers throughout the experiment. The 10-week-old female golden Syrian hamsters (LVG strain, Charles River Laboratories) were separated into groups for exposure studies with an additional sentinel group that was inoculated directly with virus.
In vivo mouse infections
Mice were lightly anesthetized with isoflurane and intranasally infected with 50 μl of virus preparation at 1 LD100—a concentration of approximately 1,000 p.f.u. for virus-packaging mutant experiments and 900 p.f.u. for LNA treatment experiments. Weights and clinical scores were assessed daily, and animals were humanely sacrificed when a clinical score of 5 was recorded (see Supplementary Table 2 for clinical score determination). Kaplan–Meier survival curves were generated using GraphPad Prism.
In vivo mouse IAV antiviral assays
‘In vivo-ready’ LNAs were custom designed and ordered from QIAGEN (formally Exiqon) and later from IDT. For intranasal delivery, in vivo-ready LNA was mixed in complexes with in vivo–JetPEI transfection reagent (Polyplus) according to the manufacturer’s protocol to the indicated final concentration in 50–75 μl of 5% glucose solution. Mice were then lightly anesthetized with isoflurane and 50–75 μl of the solution was delivered intranasally. For retro-orbital delivery, in vivo-ready LNA was mixed in complexes with in vivo–JetPEI transfection reagent (Polyplus) according to the manufacturer’s protocol to the indicated final concentration in 200 μl of 5% glucose solution. Mice were then anesthetized and the solution was delivered by retro-orbital injection. OSLT (Sigma-Aldrich, catalog no. SML1606) was prepared in sterile water and administered to mice at a dose of 10 mg kg−1 twice daily by oral gavage (totaling 20 mg kg−1 d−1), with 8 h between dosing intervals.
Prevention of SARS-CoV-2 transmission in Syrian hamsters
Infection of donor sentinel hamsters (nontreated): 6-week-old WT golden Syrian hamsters were intranasally infected with 1 × 104.3 CCID50 (cell culture infectious dose 50%) of SARS-CoV-2 (USA_WA1/2020 strain) in a 100-µl volume. LNA pretreatment group: 6-week-old golden Syrian hamsters (n = 5) were pretreated by intranasal instillation with a 200-µl volume containing 100 µg of LNA-12.8 on day −1 and day 0 before exposure to the SARS-CoV-2-infected sentinel hamsters. Vehicle-treated hamsters (n = 4) received PBS by intranasal nebulization. LNA- and vehicle-treated hamsters were co-housed and exposed to the infected sentinels for 2 h per d for 3 consecutive days. Then, 4 d after the initial exposure, the lungs were harvested and virus titers were determined by CCID50 in triplicate. Statistical analysis was performed using an unpaired Student’s t-test with GraphPad Prism 9 software.
LNA design and preparation
Oligonucleotides containing LNAs were custom synthesized by Exiqon and later by IDT. Capitalized letters denote LNA. Lower-case letters denote typical (nonlocked) DNA nucleotides. All oligonucleotides contain phosphorothioate internucleoside linkages. LNA8 and -9 were designed as LNA gapmers to contain a stretch of six or seven DNA nucleotides optimized for RNase-H recruitment. Sequences of all LNAs are shown below:
LNA1: 5′-AccAaaAGaaT-3′
LNA2: 5′-TggCcATcaaT-3′
LNA3: 5′-TagCAtActtA-3′
LNA4: 5′-CCAAAAGA-3′
LNA5: 5′-CATACTTA-3′
LNA6: 5′-CagaCaCGaCCaaAA-3′
LNA7: 5′-TAcTtaCTgaCagCC-3′
LNA8: 5′-AGAcacgaccaaAAG-3′
LNA9: 5′-TACTtactgacaGCC-3′
LNA14: 5′-CGACcaaaagaATTC-3′
Scr. LNA (negative control): 5′-AACACGTCTATACGC-3′
SARS-CoV-2-directed LNAs:
LNA-12.8: 5′-AGGAagttgtagCACG-3′
LNA-14.3: 5′-GCTctccatcttaCCT-3′
In vitro LNA antiviral assays
For all experiments, LNAs were reconstituted in RNase-free water at 100 μM stock solutions, aliquoted and stored at −20 °C before single use. Lipofectamine 3000 (Life Technologies) was used to transfect LNA into cells at indicated concentrations per the manufacturer’s protocol.
For IAV prophylactic antiviral assays, 1 × 106 MDCK cells were plated in 6-well plates 24 h before being transfected with the indicated LNA. Cells were then infected at the indicated time points with 0.01 MOI of PR8 (H1N1) or HK68 (H3N2) virus. For post-infection therapeutic assessment, MDCK cells were infected with PR8 or HK68 before LNA transfection as described above. Then 48 h post-infection, supernatant was collected and the viral titer was determined by plaque assay in triplicate.
For SARS-CoV-2-Nluc assays: 1 d before transfection, Huh-7 or ACE2-TMPRSS2-Huh-7.5 was plated on 96-well clear-bottomed plates to 60–70% confluency at the time of treatment with the LNA ASOs LNA-12.8, LNA-14.3 or Scr. LNA. Lipofectamine 3000 (Life Technologies) was used to transfect LNA ASOs into cells at 25 nM or 100 nM final concentration, according to the manufacturer’s protocol. Cells were then infected with SARS-CoV-2 reporter virus expressing nanoluciferase (SARS-CoV-2-Nluc) at an MOI of 0.3 for 1 h, after which the virus was removed and fresh medium was added. Recombinant SARS-CoV-2-Nluc is a fully replicating virus in which ORF7 has been deleted and replaced with Nluc. Thus, the measurement of Nluc expression is a surrogate marker of virus replication enabling the screening of antiviral compounds. A nucleoside analog β-d–N4-hydroxycytidine, EIDD-1931, with potent activity against SARS-CoV-2, was included as a positive control. A DMSO control was included as a mock-treated, negative control. Data were graphed and analyzed in Prism v.8 and v.9 by GraphPad. Statistical analysis of the data from each cell type was computed as an ordinary one-way analysis of variance (ANOVA) using Dunnett’s multiple comparison test against the DMSO control or Scr. LNA control from each cell type, where indicated.
SARS-CoV-2 plaque assay experiments were performed in 24-well plates: 24-well plates were seeded with 1 × 105 Vero E6 cells per well in DMEM, 10% FBS and 1× antibiotic/antimycotic 24 h before Lipofectamine 3000 transfection of LNA. Scr. LNA was included as a negative control. Then 12 h post-transfection, LNA-transfection medium was removed, and cells were infected at an MOI of 0.01 with either recombinant WT SARS-CoV-2 (PMID: 32526206) or a SARS-CoV-2 clinical isolate derived from a nasopharyngeal swab taken from a chronically infected cancer patient 7 months after the initial confirmatory COVID-19 PCR test. After adding virus inoculum, plates were incubated at 37 °C for 1 h, after which input virus was removed, wells were washed with 1 ml of medium and 1 ml of ‘infection medium’ (DMEM, 5% FBS, 1× antibiotic/antimycotic) was added. After 48 h at 37 °C, 200 µl of culture supernatant was collected and stored at −80 °C until quantitation of infectious virus by plaque assay. Briefly, Vero E6 cells were seeded at 500,000 cells per well per 2 ml in 6-well plates. After 24 h, samples were thawed at room temperature and serially diluted in PBS. Medium was removed from the six-well plates and serial dilutions were added to the plate, incubated for 1 h at 37°C and then overlaid with DMEM, 5% FBS, 1× antibiotic/antimycotic and 0.9% agarose. After 72 h, neutral red stain was added to each plate, incubated for 3 h and plaques were counted. The number of plaque-forming units (p.f.u.) per milliliter was generated using the following formula: p.f.u. ml−1 = number of plaques × serial dilution factor × 5.
LNA treatment and IAV-packaging efficiency determination
Briefly, T75 flasks of 80% confluent MDCK cells were transfected with 100 nM of Scr. LNA, LNA9 or mock untreated by Lipofectamine 3000 transfection, according to the manufacturer’s protocol. Then 12 h post-transfection, cells were infected with 0.01 MOI of WT TC-adapted PR8 virus. After 1 h, virus was removed and the cells were washed with PBS; 48-h post-infection supernatants were collected and RNA was isolated as described in isolation of packaged vRNAs and assay methods.
In vitro drug selection
LNA9 selection: 80–90% confluent MDCK cells in 12-well plates were transfected in duplicate with a starting concentration of 0.01 nM (~½EC50) LNA9 for passage 1 by Lipofectamine transfection (see above). Then 12 h post-transfection, cells were washed with PBS and infected with an MOI of 0.01 of WT PR8 virus. After 1-h incubation at 37 °C, cells were washed and virus growth medium was added. Cells were incubated until 50% cytopathic effect (CPE) was evident (48–72 h). Virus supernatant was harvested, low-speed centrifuge clarified, aliquoted, plaque titered and stored at −80 °C. The virus supernatant was then continuously serially passaged in the presence of escalating concentrations of LNA9 (0.01 nM to 100 nM). If no CPE were evident, the drug concentration was lowered and the added virus concentration increased until 50% CPE occurred. OSLT selection: confluent MDCK cells in 12-well plates were infected with an MOI of 0.01 of PR8 virus. After adsorption for 1 h, cells were washed with PBS and OSLT (Sigma-Aldrich, catalog no. Y0001340) was added to the virus growth medium at a starting concentration of 1 nM (~½EC50). Drug selection proceeded as described above, with escalating concentrations of OSLT (0.01 nM to 250 μM) at each subsequent passage.
EC50 determination
For LNA9, the EC50 was defined as the concentration of drug effective in reducing the percentage of virus titer to 50% of that for the no-drug control. In brief, the EC50 was determined by seeding 5 × 105 MDCK cells in each well of a 12-well plate and incubating overnight at 37 °C under 5% CO2. Cells were then transfected with LNA9 as described above at concentrations from 0.01 nM to 10 μM. Plates were incubated at 37 °C for 12 h before infection with 0.01 MOI of WT PR8, serially passaged LNA-treated virus, WSN33 WT or WSN33 H275Y NAI-resistant virus. Then 48 h post-infection, supernatants were collected, centrifuge clarified, aliquoted and stored at −80 °C. The viral titer for each drug dilution was performed by plaque assay in duplicate. The EC50 was the concentration of LNA9 yielding a percentage titer of 50% of that without drug.
For OSLT, the EC50 was defined as the concentration of drug reducing the total percentage of plaques to 50% of that for the no-drug control, determined by plaque reduction assay1. Briefly, confluent MDCK cells in 12-well plates were infected with approximately 100 p.f.u. of WT PR8, serially passaged OSLT-treated virus, WSN33 WT or WSN33 H275Y NAI-resistant virus and incubated for 1 h at 37 °C. Cells were then washed with PBS and a 50:50 mix of 1% agarose: 2× virus growth DMEM containing varying concentrations of drug (0.1 nM to 1 mM) was added to the cells. Plates were harvested 72 h later, stained with Crystal Violet and plaques were counted. The EC50 was the concentration of OSLT reducing the total percentage of plaques to 50% of that without drug. All results were plotted in GraphPad Prism to generate EC50 curves.
In vitro transcription of full-length IAV vRNA
For each WT isolate (PR8, 1918, VN1203, NY470, NY312 and CA09) and PR8 packaging mutant clones, PB2 cDNA was amplified from plasmid-using, segment-specific primers under a T7 promoter. Amplified cDNA was gel purified using an Invitrogen DNA gel kit. The vRNAs were then produced by in vitro transcription, using T7-MEGAscript kit. The vRNAs for SHAPE were purified by MEGAclear (Thermo Fisher Scientific, catalog no. AM1908) with purity and length verified by capillary electrophoresis.
The single fluorophore-SHAPE 1D analysis of full-length IAV vRNA
In vitro transcribed PB2 vRNA was folded (100 mM NaCl, 2.5 mM MgCl2, 65 °C for 1 min, 5-min cooling at room temperature, 37 °C for 20–30 min) in 100 mM Hepes, pH 8. The 2′-acylation with N-methylisatoic anhydride18 and RT primer extension were performed at 45 °C for 1 min, 52 °C for 25 min and 65 °C for 5 min, as previously described44. 6-Carboxyfluorescein (6FAM) was used for all labeled primers (primer sequences available on request). Exceptions to these protocols were as follows: (1) RNA purification after acylation was performed using RNA C&C columns (Zymo Research), rather than ethanol precipitation; (2) before and after SHAPE primer buffer was added, the mixture was placed at room temperature for 2–5 min, which enhanced RT yields significantly; (3) DNA purification was performed using Sephadex G-50 size exclusion resin in 96-well format, then concentrated by vacuum centrifugation, resulting in a more significant removal of primer; and (4) 2 pmol of RNA was used in ddGTP (2′,3′-dideoxyguanosine-5′-triphosphate) RNA-sequencing reactions.
The ABI 3100 Genetic Analyzer (50-cm capillaries filled with POP-6 matrix) was set with the following parameters: voltage 15 kV, T = 60 °C, injection time = 15 s. The GeneScan program was used to acquire the data for each sample, which consisted of purified DNA resuspended in 9.75 μl of Hi-Di-Formamide, to which 0.25 μl of ROX500 internal size standard (ABI catalog no. 602912) was added. PeakScanner parameters were set to the following parameters: smoothing=none; window size=25; size calling=local southern; baseline window=51; peak threshold=15. Fragments 250 and 340 were computationally excluded from the ROX500 standard45. The data from PeakScanner were then processed into SHAPE data by using FAST (fast analysis of SHAPE traces), a customized algorithm developed in our lab19. FAST automatically corrects for signal differences due to handling errors, adjusts for signal decay and converts fragment length to nucleotide position, using a ddGTP ladder as an external sizing standard and the local Southern blotting method5,19. This algorithm embedded in the RNAstructure program is freely available at http://med.stanford.edu/glennlab/download.html.
RNAstructure parameters: slope and intercept parameters of 2.6 and −0.8 kcal mol−1 were initially tried, as suggested46; however, we found that smaller intercepts closer to 0.0 kcal mol−1 (for example, ~−0.3) produced fewer less optimal structures (within a maximum energy difference of 10%). We speculate that this minor parameter difference may be due to the precise fitting achieved between experimental and control datasets by the automated FAST algorithm. FAST was written in ANSI C/C++ and is integrated into RNAstructure with FAST, which requires MFC (Microsoft Foundation Classes). RNA structures were drawn and colored using RNAViz 2 (ref. 47) and finalized in Adobe Illustrator.
IAV PSL2 construct design, RNA synthesis and chemical modification for M2 experiments
Double-stranded DNA templates were prepared by PCR assembly of DNA oligomers designed by an automated MATLAB script as previously described (available at https://primerize.stanford.edu)48. Constructs for M2 include all single mutants to the Watson–Crick counterpart. Compensatory mutants for M2R were designed based on basepairing in the proposed secondary structure22. In vitro transcription reactions, RNA purification and quantification steps were as described previously48. One-dimensional (1D) chemical mapping, M2 and M2R were carried out in the 96-well format as described previously48,49,50. Briefly, RNA was heated up and cooled to remove secondary structure heterogeneity, then folded properly and incubated with SHAPE reagent (5 mg ml−1 of 1-methyl-7-nitroisatoic anhydride (1M7))51; modification reaction was quenched and RNA was recovered by poly(dT) magnetic beads (Ambion) and FAM-labeled Tail2-A20 primer; RNA was washed by 70% ethanol twice and resuspended in double-distilled water (ddH2O), followed by RT to cDNA and heated NaOH treatment to remove RNA. The final cDNA library was recovered by magnetic bead separation, rinsed, eluted in Hi-Di-Formamide (Applied Biosystems) with ROX350 ladder and loaded to a capillary electrophoresis sequencer (ABI 3100). Data processing, structural modeling and data deposition: the HiTRACE software package v.2.0 was used to analyze CE data (both MATLAB toolbox and web server available52,53). Trace alignment, baseline subtraction, sequence assignment, profile fitting, attenuation correction and normalization were accomplished as previously described54,55. Sequence assignment was accomplished manually with verification from sequencing ladders. Data-driven secondary structure models were obtained using the Fold program of the RNAstructure package v.5.4 (ref. 56) with pseudo-energy slope and intercept parameters of 2.6 kcal mol−1 and –0.8 kcal mol−1. Two-dimensional z-score matrices for M2 datasets and helix-wise bootstrapping confidence values were calculated as described previously22,48. The z-score matrices were used as basepair-wise, pseudo-free energies with a slope and intercept of 1.0 kcal mol−1 and 0 kcal mol−1. Secondary structure images were generated by VARNA57. These chemical-mapping datasets, including 1D mapping, M2 and M2R have been deposited at the RNA Mapping DataBase (RMDB: http://rmdb.stanford.edu)58, accession nos.: PSL2IAV_1M7_0001, PSL2IAV_RSQ_0001.
SHAPE analysis of LNA-targeted IAV vRNA
A truncated DNA template of PR8 virus segment PB2 containing nucleotides 1–88 was prepared by PCR assembly of DNA oligomers, and in vitro transcription reactions, RNA purification and quantification steps were as described previously48. The 1D SHAPE chemical mapping was performed in a 96-well plate format as described above, with the following exception: once RNA was denatured and refolded as described, 100 nM of each prepared LNA was added to the folded RNA and incubated with 5 mg ml−1 of SHAPE reagent 1M7. Modification quenching, RNA recovery, re-suspension, RT, cDNA-sequencing and data processing were performed as described50.
SHAPE 1D analysis of nontreated and LNA-treated SARS-COV-2 RNA
RNA was folded (0.5 M Na Hepes, pH 8; 90 °C for 3 min, 12-min cooling at room temperature, 50 °C for 20 min, 12-min cooling at room temperate) in 100 mM MgCl2 with or without LNA and with or without SHAPE reagent 1M7 modification. RNA was purified and quenched with magnetic beads (0.5 M 2-(N-morpholino)ethanesulfonic acid sodium salt, pH 6, FAM-A20 tail 2 primer, 5 M NaCl and Ampure beads) and reverse transcribed at 48 °C for 40 min, followed by a 0.4 M NaOH acid quench to improve signal intensity. The resulting cDNA was resuspended in ROX Hi-Di-Formamide and diluted for capillary electrophoresis analysis. As an internal control, the RNA was created with GAGUA hairpins in 5′- and 3′-termini designed to be reactive in the presence of 1M7. The results were analyzed using the HiTRACE method52 and standardized by the Kladwang et al. method55.
PBMCs and splenocyte isolation
Whole blood was collected from mice into heparinized tubes. The whole blood was overlaid on top Ficoll-Paque medium and centrifuged at 400g for 40 min at room temperature. The top layer containing plasma and platelets was removed and the peripheral blood mononuclear cells (PBMCs) at the interphase of the Ficoll layer were collected. The mononuclear cells were diluted in PBS and pelleted by centrifugation at 500g for 15 min after an additional wash with PBS. The pellet was suspended in PBS. Splenocytes were isolated by manually grinding the spleen over a 40-μm cell strainer. The cells were transferred several times through the strainer and processed further as described above. TruStain fcX (anti-mouse CD16/32) antibody specific for FcyR III/II (1 μg per 1 × 106 cells) was used to block nonspecific staining, followed by staining with Zombie Aqua viability kit (BioLegend). Antibody staining was performed by mixing the cells with an antibody mix containing BD Horizon PE-CF594 Rat Anti-Mouse CD45 (BD Biosciences), PE/Cy7 anti-mouse CD3 (BioLegend), APC/Cy7 anti-mouse CD8a (BioLegend) and H-2Kd Influenza HA Tetramer-IYSTVASSL-PE (MBL International). All antibodies were diluted at a ratio of 2 ml of each antibody per 1 × 106 cells and HA tetramer was the diluter at a ratio of 5 ml per 1 × 106 cells. Splenocytes were isolated from a mouse spleen by immersing the spleen in Hanks’ balanced salt solution (HBSS) with 10% FBS and washing with HBSS to remove blood. The spleen was placed in a 40-mm cell strainer and the tissue was mashed with a syringe plunger to break down the tissue and dislodge the cells. After complete disruption of the organ, the cells were run through the strainer into a conical tube. The cell suspension was pelleted by centrifugation at 350g for 10 min at 4 °C. The pellet was resuspended in water for 20 s to disrupt red blood cells followed by addition of 2× PBS solution. The cells were pelleted and subjected to staining as described for PBMCs above.
Flow cytometry
After staining, the cells were fixed and subjected to flow cytometry using a BD LSR II flow cytometer (BD Biosciences) equipped with 488-nm, 405-nm, 640-nm and 532-nm lasers. Data were collected using BD FACSDiva software (BD Biosciences) and analyzed using FlowJo software (TreeStar). The gating strategy for positive tetramer cells was as follows: forward scatter area (FSC)-H/FSC-A gate used to collect cells and a gate for live cells was then generated. The live cells were gated for CD3+/CD45+ cells and the positive cells were gated to determine the CD8+/HA-tetramer+ cells. Acquired data was analyzed using a FlowJo software (TreeStar).
LNA effect on IFN and NF-kB pathways
THP1-Dual or A549-Dual cells (InvivoGen) were transfected in triplicate with LNA9, LNA14 or derivatives thereof, or poly(I:C) (R&D Systems) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Treatment with recombinant human tumor necrosis factor α (hTNF-α; R&D Systems), recombinant human IFN (hIFN-γ; R&D Systems), recombinant hIFN-α (R&D Systems) or lipopolysaccharide (LPS) from O111:B4 Escherichia coli (Sigma-Aldrich) was used as a positive control for activation of the two pathways. Supernatant from the cultures were collected 24–32 h post-transfection/treatment to measure luciferase signal or secreted alkaline phosphatase activity as indicators of IFN pathway stimulation or NF-κB pathway stimulation, respectively. Results were normalized to reflect fold induction relative to NT cells.
IL-6 and TNF-α ELISA
Supernatants from THP1-Dual cells (InvivoGen) were transfected in triplicate with LNA9, LNA14 or poly(I:C) (R&D Systems). Treatment with recombinant hIFN-γ (R&D Systems), recombinant hIFN-α (R&D Systems) or LPS from O111:B4 E. coli (Sigma-Aldrich) was used as a positive control to cause induction of interleukin (IL)-6 or TNF-α secretion. Supernatants were collected 24 h post-transfection/treatment and assayed for TNF-α and IL-6 concentration by ELISA (Thermo Fisher Scientific) according to the ELISA kit protocol.
Statistical analyses
We expressed the data as the mean ± s.d. or mean ± s.e.m. where indicated. We used Student’s t-test (to compare two samples) or ANOVA (to compare multiple samples) as analyzed by GraphPad Prism (v.8 and v.9) for statistical analysis. We performed Kaplan–Meier log(rank) tests for survival analyses. We considered all P values >0.05 not to be significant.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
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