Journal: Molecular cell
Article Title: Structural basis for OAS2 regulation and its antiviral function.
doi: 10.1016/j.molcel.2025.05.001
Figure Lengend Snippet: Figure 4. OAS2 dimerization and localization to the Golgi membrane via myristoylation are required for activation (A) SDS-PAGE (top) and BN-PAGE (bottom) of OAS2 constructs transiently expressed in HEK293T cells and purified His-tagged OAS2 WT dimer. Zinc coor- dination is depicted on the right. SDS-PAGE was cut to remove a lane with marker. (B) OAS2 activity in HEK293T cells measured with 2′–5′OA biosensor 24 h after poly I:C transfection, showing higher activity of dimeric OAS2 than the monomeric mutants. (C) Immunofluorescence Airyscan microscopy of endogenous OAS2 localization in primary HFF cells stimulated with IFN-α and stained for OAS2 (green), Golgi marker GM130 (magenta), and DAPI (blue). Scale bars represent 20 μm and 2 μm for merge zoom. (D) As in (C) for A549 OAS2 KO cells reconstituted with doxycycline-inducible OAS2 constructs treated with doxycycline. Scale bars represent 10 μm. (E) Quantification of colocalization of OAS2 constructs and Golgi marker GM130 from (D). Lines represent means from analyzed individual cells (dots). Student’s t test with Welch’s correction, ***p ≤0.001. (F) Analysis of OAS2 activity in cells as in (B) for OAS2 wild type and mutants with different localization showing that Golgi targeting is essential for the activity. (G) Surface view of OAS2 dimer, with each monomer depicted in gray and blue. Close-up shows dimer interface interactions of S150 and D153. (H) SDS-PAGE and BN-PAGE analysis as in (A). (I) In vitro chromogenic activity assay with 200 nM OAS2 wild type and OAS2 S150A D153A with 23, 44, and 82 bp dsRNA (200 nM) (mean ± SD of n = 3). (J) Analysis of OAS2 activity in cells as in (B) for OAS2 wild type and DI-DII interaction mutant S150A D153A. In (B), (F), and (J) bars represent means ± SD of four (B), five (F), and six (J) independent replicates (dots). Paired t test: ns p > 0.05, * p ≤0.05, ** p ≤0.01, *** p ≤ 0.001, **** p ≤0.0001. See also Figures S5 and S6.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-OAS2 R&D Systems Cat#MAB1925-SP; RRID:AB_215616 anti-OAS2 Proteintech Cat#19279-1-AP, lot# 00113570; RRID:AB_10642832 anti-OAS1 Cell Signaling Cat#14498S; RRID:AB_2798498 anti-GM130 Thermo Fisher Scientific Cat#703794; RRID:AB_2848241 anti-GM130 Proteintech Cat#66662-1-Ig; RRID:AB_2882017 anti-calnexin R&D Systems Cat#NB100-1965SS; RRID:AB_10002123 anti-giantin R&D Systems Cat#NBP1-91937; RRID:AB_11024007 anti-TGN46 R&D Systems Cat#NBP1-49643SS; RRID:AB_10011761 anti-Golgi 58K Sigma-Aldrich Cat#G2404; RRID: AB_477002 anti-dsRNA J2 SCICONS Cat#1qq0010500; RRID: AB_2651015 anti-OAS3 Proteintech Cat#21915-1-AP RRID:AB_2876880 anti-GAPDH Santa cruz Cat#sc-47724; RRID:AB_627678 anti-FLAG-M2-HRP Sigma Aldrich Cat#A8592; RRID:AB_439702 anti-RNase L Santa cruz Cat#sc-74405; RRID:AB_2181661 anti-OAS1 Proteintech Cat#14955-1-AP; RRID:AB_2158292 anti-GFP Abcam Cat# ab290; RRID: AB_2313768 anti-mouse AF488 Invitrogen Cat#A11029; RRID: AB_2534088 anti-rabbit AF647 Thermo Fisher Cat#A31573; RRID: AB_2536183 anti-rabbit AF488 Cell Signaling Cat#4412S; RRID: AB_1904025 anti-mouse AF647 Thermo Fisher Cat#A31571; RRID: AB_162542 anti-rabbit AF488 Invitrogen Cat# A-11008; RRID: AB_143165 Anti-mouse AF594 Invitrogen Cat#A11032; RRID: AB_2534091 Bacterial and virus strains E. coli Rosetta Expression Systems N/A E. coli STBL3 Andreas Pichlmair N/A E. coli DH5alpha Andreas Pichlmair N/A E. coli DH10alphaMultiBac Expression Systems N/A BUNV wt Friedemann Weber N/A EMCV Andreas Pichlmair, Pichlmair et al.85 N/A HSV1(17+)Lox-GFP Beate Sodeik N/A LACV (rec wt) Friedemann Weber N/A THOV-ML- Andreas Pichlmair, Haas et al.86 N/A VACV-V300-GFP Joachim Bugert N/A VSV-AV3-GFP (Indiana) Andreas Pichlmair lab, Pichlmair et al.87 N/A YFV (YF-17D)-Venus Simon Rothenfußer, Santos-Peral et al.88 N/A YFV (YF-17D) Simon Rothenfußer, Santos-Peral et al.88 N/A ZIKV H/PF/2013 Andreas Pichlmair lab, Scaturro et al.89 N/A IAV SC35M PB2 2A GFP Martin Schwemmle N/A RVFV-GFP Friedemann Weber, Habjan et al.90 N/A HRV16 ATCC Cat# VR-283 HCoV-OC43 ATCC Cat# CR-1558TM NL63 Lia van der Hoek N/A (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 85, 1–18.e1–e13, June 5, 2025 e1
Techniques: Membrane, Activation Assay, SDS Page, Construct, Purification, Marker, Activity Assay, Transfection, Immunofluorescence, Microscopy, Staining, In Vitro, Mutagenesis
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![Figure 6. OAS2 loss of function is associated with immune dysregulation with ANCA vasculitis (A) Patient’s serum levels of inflammatory markers C-reactive protein (CRP; [<0.5 mg/dl] and soluble interleukin-2 receptor (sIL-2R; [158–613 U/ml]), and anti- bodies against myeloperoxidase (MPO; [<20 U/ml]). Gray bars indicate normal ranges. (B) Renal biopsy with hematoxylin and eosin stain, 20x magnification. (C) Pedigree of family with OAS2 F524L mutation. (D) Electropherograms showing de novo heterozygous OAS2 variant (c.1572C>G; p.Phe524Leu) by Sanger sequencing. (E) Multiple sequence alignment of human OAS proteins and selected human NTases showing high conservation for F524 (yellow star). (F) Structure of the OAS2 dimer with close-up view highlighting the amino acid F524. (G) Expression levels of OAS2 in wild-type (WT) and patient fibroblasts treated with IFN-α (n = 2). (H) OAS2 immunofluorescence staining in WT and patient fibroblasts treated with IFN-α. Scale bars represent 50 μm. (I) Quantification of OAS2 mean fluorescence intensity from (H). Lines represent means from measurements of individual cells (dots). Student’s t test with Welch’s correction, **** p ≤0.0001. (J) RNA pico-chip analysis of total RNA isolated from wild-type (WT) and patient-derived fibroblast cell lines from control or poly I:C transfection.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_2389/pm40412389/pm40412389__page11_image1.jpg)