Fig. 4: LNAs targeting PSL2 RNA structure display potent antiviral activity in vitro.
a, Regions of PSL2 targeted by indicated LNAs. b, Antiviral screen of LNAs transfected into MDCK cells and infected 4 h later with PR8 (H1N1) or A/Hong Kong/8/68 (H3N2) virus (0.01 MOI) and viral titers determined 48 h post-infection (n = 3). Statistics performed by unpaired, ordinary one-way ANOVA (PR8 and HK68) with Dunnett’s multiple comparison test. c, Antiviral efficacy as a function of time of LNA addition (n = 3), analyzed as in b. Statistics are by two-way ANOVA with Dunnett’s multiple comparison test against nontreated +Lipo3k (NT). d, PB2 vRNA (PR8) packaging efficiency of viruses treated with 100 nM LNA9 or Scr. LNA control. Values are given as a percentage of PB2 vRNA packaging in comparison to NT WT PR8 virus; readout by qPCR. The results are from two biological replicates (n = 2), assays performed in technical triplicates. e, LNA9 efficacy against multiple IAV strains in MDCK cells pretreated with 100 nM of indicated LNAs, analyzed as in b. The statistics are as described in c against the NT control. f, In vitro selection for drug resistance to LNA9 with escalating concentrations of LNA and the sensitivity of passaged virus in response to drug treatment. The EC50 values are determined at the indicated passage (P) numbers. The results are expressed as a percentage of NT virus titer. g, In vitro selection of PR8 virus selected with OSLT. OSLT-treated PR8 virus and drug sensitivity were determined by plaque reduction assay. The number of viral plaques with each drug concentration was normalized against the NT control to determine the EC50. h,i, In vitro sensitivity of WT WSN33 (H1N1) and NAI-resistant (WSN H275Y NA mutant) virus to LNA9 (h) or OSLT (i). The EC50 values were computed using a nonlinear regression model with variable slope. Statistics for all graphs performed in GraphPad Prism 9 software. All error bars represent mean ± s.d. *Statistical P value is indicated in each panel.
