Fig 2: Expression pattern of the OAS–RNase L pathway genes and their role in SARS-CoV-2 restriction.(A and B) Relative OAS1, OAS2, OAS3, and RNASEL mRNA levels measured by bulk RNA-seq (A) or RT-qPCR (B), in various cell types. hPSC, human pluripotent stem cell; ClassMonocytes, classical monocytes; NClassMonocytes, nonclassical monocytes; MDM, monocyte-derived macrophages; MDDC, monocyte-derived dendritic cells; Log2RC, log2 read count. (C and D) Immunoblot of the indicated proteins (C) and immunofluorescence (IF) of SARS-CoV-2 nucleocapsid (N) protein (D) in A549+ACE2/TMPRSS2 cells with and without knockout (KO) of OAS1, OAS2, or RNase L. IF analysis for N protein was performed 24 hours after infection with various dilutions of SARS-CoV-2. Dilution factors of ¼, ½, and 1 correspond to MOI values of 0.0002, 0.0005, and 0.001, respectively. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NI, noninfected. (E and F) Immunoblot of the indicated proteins (E) and IF analysis for the SARS-CoV-2 N protein (F) in SV40-fibroblasts +ACE2 from healthy controls (Ctrl1 and Ctrl2), patients with OAS-RNASEL mutations (P1, P3, P4, and P5), and a previously reported patient with complete IFNAR1 deficiency (IFNAR1-/-). IF analysis for N protein was performed at various time points after infection at a MOI of 0.08. (G and H) Immunoblot of the indicated proteins (G) and IF analysis for the SARS-CoV-2 N protein (H) in THP-1 cells with and without KO of OAS1, OAS2, or RNase L. IF analyses for N protein were performed in PMA-primed THP-1 cells 24 hours after infection with various dilutions of SARS-CoV-2. Dilution factors of ¼, ½, and 1 correspond to MOI values of 0.012, 0.025, and 0.05, respectively. WT A549+ACE2/TMPRSS2 cells were included as a positive control for SARS-CoV-2 infection. The data points are means ± SEM from three [(D) and (F)] or means from two [(B) and (H)] independent experiments with three to six technical replicates per experiment. Statistical analyses were performed as described in the methods. ns, not significant; *P < 0.05, **P < 0.01.

Fig 3: Biallelic OAS1, OAS2, and RNASEL variants in patients with MIS-C.(A) Family pedigrees with allele segregation. Mutant, “MT” in red; wild-type, “WT” in black. (B to D) Functional assays for WT and mutant OAS1 (B), OAS2 (C), and RNase L (D). Variants for which homozygotes or compound heterozygotes were present in our MIS-C cohort were tested. (Upper panels) RNase L–mediated cleavage of rRNA in a cell-free system based on transfected HeLa M cells. (Lower panels) Immunoblots of the indicated proteins. EV, empty vector. Arrows indicate degraded rRNA species. OAS2 variants (C) were tested under two different sets of conditions (see methods). The results shown in (B) to (D) are representative of three independent experiments. (E and F) FRET assay of 2–5A synthesized in response to poly(I:C) stimulation by WT and MT OAS1 (E) or OAS2 (F). RFU, relative fluorescence units. The data shown are the means ± SEM of six biological replicates. Statistical analysis was performed as described in the methods. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (G) Concentrations of various cytokines in plasma samples from OAS–RNase L–deficient patients during MIS-C (P1, P2, and P5); comparison with those of healthy controls (HC), pediatric (pC-19) or adult COVID-19 pneumonia (aC-19) patients, typical Kawasaki disease patients (KD), other MIS-C patients with no known genetic etiology (MIS-C), and patients with toxic shock syndrome (TSS). (H) PCA of gene expression quantified by whole-blood bulk RNA-seq for P1 and P2 during MIS-C relative to pediatric controls (pCtrls), previously published MIS-C patients, and a pediatric patient with mild COVID-19 (pC-19). (I) Relative levels of TRBV 11–2 (encoding Vß21.3) RNA in blood samples from P1, P2, and P5 during MIS-C, relative to other MIS-C patients, adults with mild or severe COVID-19 (mild aC-19, sev aC-19), and healthy controls. (J to L) CADD-MAF graph of OAS1 (J), OAS2 (K), and RNASEL (L) variants for which homozygotes are reported in gnomAD and/or found in our MIS-C cohort. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Fig 4: Exaggerated inflammatory responses of OAS–RNase L-deficient THP-1 cells.(A) Concentrations of various cytokines in the supernatant of OAS1 KO, OAS2 KO, RNase L KO, or parental THP-1 cells (upper panels) or PMA-primed THP-1 cells (lower panels) treated as indicated for 24 hours. (B) IFN-?1 and IL-6 concentrations in the supernatant of RNase L KO THP-1 cells transduced with the WT or P5’s variant RNASEL cDNA, or empty vector (EV), and treated as indicated for 24 hours. On the right, RNase L protein levels, as assessed by immunoblotting. NT, not transfected. (C) IFN-?1 and IL-6 concentrations in the supernatant of parental, RIG-I KO, MDA5 KO, or MAVS KO THP-1 cells with or without (WT sh-ctrl) RNase L knockdown (KDn), treated as indicated for 24 hours. (D) IFN-?1 and IFN-ß concentrations in the supernatant of parental or RNase L KO THP-1 cells, treated as indicated for 24 hours. (E) Immunoblot of phosphorylated P65 and IRF3 in parental and RNase L KO THP-1 cells treated as indicated. The results shown are representative of two independent experiments. (F) IFN-?1 and IL-6 concentrations in the supernatant of parental, OAS1 KO, or RNase L KO THP-1 cells treated as indicated for 24 hours. (G) IFN-?1 and IL-6 concentrations in WT THP-1 cells treated as indicated for 24 hours. In (A) to (D), (F), and (G), the data points are means ± SEM from three to five independent experiments with one to two technical replicates per experiment. Statistical analysis was performed as described in the methods. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, nonstimulated; Lipo, lipofectamine only; poly(I:C), extracellularly added poly(I:C); poly(I:C)+Lipo, intracellular poly(I:C) in the presence of lipofectamine; 2–5A+Lipo, intracellular 2–5A in the presence of lipofectamine; 2–5A+poly(I:C)+Lipo, intracellular poly(I:C) in addition to intracellular 2–5A.

Fig 5: RNaseL expression pattern in cells of NVU. Expression of RNaseL in primary human brain pericytes, astrocytes, EC, immortalized human microglial cells, and SH-SY5Y neuroblastoma cell line as measured by immunoblotting (A). Pericytes were either mock-infected or infected as in Fig. 6, and mRNA and protein expression of RNaseL was measured by qPCR and immunoblotting (B). Pericytes were transfected with ocln overexpressing vector PCMV3-OCLN or with PCMV3 control vector, and the expression of RNaseL was evaluated by qPCR and immunoblotting (C). GAPDH was used as a housekeeping gene and loading control. Values are mean ± SEM. ****p<0.0001, ***p=0.0002, **p=0.003, *p<0.05, n=4–6 independent samples per group