Fig. 3: Construction of low-immunogenicity triple-mutant huNoV saRNA.

A, B Predicted binding free energy (BE) and interaction patterns between huNoV RdRp(pro) NT and four NTPs by Autodock molecular docking (100 predictions each). C VPg nucleosidation using huNoV RdRp-WT or mutants (ΔT84, ΔK146, and ΔY151), with the ratios of nucleosidylated VPg species quantified by high-resolution HPLC. D VPg nucleosidation using RdRp-WT or additional mutants (V82G, A1G/P2Q, A1G/P2Q/V82G), with HPLC-derived peak-area ratios displayed. E, F Representative dot blotting and automated Western blotting results from three independent experiments detecting dsRNA and VP1/VP2 in Huh-7 cells transfected with huNoV gRNA saRNA-LNPs carrying the c.3031C > G, c.3034C > A, or c.3274T > G mutations; Ponceau S and GAPDH served as loading controls. G, H qPCR and ELISA analyses comparing ISG induction (ifit1, Mx2, infa1, and infb1) and cytokine release (IFN-α, IFN-β, and CXCL10) between WT and triM huNoV gRNA saRNA. I Schematic of the huNoV egfp triM-saRNA design: antisense initiation/termination sequences flanking the GOI antisense strand, an upstream replication termination signal (RTS), and 5′ cap1 ensuring ORF1 translation. The production of VPg-pU, triple-mutant RdRp, and 2C-L enables negative-strand synthesis and RTS-mediated release of VPg-linked GOI positive strands for translation. J Representative dot blot images from three independent experiments detecting dsRNA in lysate supernatants of 293T cells transfected with huNoV EGFP triM-saRNA-LNPs or VEEV EGFP-saRNA-LNPs. K, L qPCR and fluorescence microplate quantification of EGFP mRNA and EGFP protein expression over time following transfection of 293T cells with saRNA-LNPs. M Representative automated Western blot images from three experiments showing PKR dimerization, phosphorylation of PKR (Thr446/451) and eIF2α (Ser51), and induction of Rig-I and MDA5 after transfection with saRNA-LNPs. N, O qPCR and ELISA comparing ISG expression and cytokine release between huNoV egfp triM-saRNA and VEEV egfp-saRNA. All cell-based experiments were performed independently three times. All data are expressed as mean ± s.d. P values were calculated using one-way ANOVA followed by Tukey’s HSD post-hoc test (C, n = 5; D, n = 5), two-way ANOVA followed by Sidak’s post-hoc multiple-comparisons test (L, n = 5; K, n = 5), and unpaired two-tailed Student’s t test (G, n = 6; H, n = 6; N, n = 6; O, n = 6). Exact p-values are indicated within the figure. Source data are provided as a Source Data file.