Fig 2: Effect of STT3B on NS4B glycosylation and synthesis. (A) 293T cell pools expressing Cas9 and sgRNAs targeting the indicated genes were transiently transfected to express NS4B-HA. Lysates were resolved by SDS-PAGE and visualized by Western blotting for the indicated proteins. Numbers to the right of the immunoblots in all panels indicate molecular mass in kDa. (B) Huh7.5.1 cell pools expressing Cas9 and sgRNAs targeting the indicated genes were transiently transfected to express NS4B-V5 (six leftmost lanes) or were mock transfected (rightmost lane). The lysates were subjected to PNGase F treatment (+) in the middle lanes to remove N-linked glycans from proteins. Proteins were resolved by SDS-PAGE and visualized by Western blotting for the indicated proteins. The black arrow to the right of the gel indicates a nonspecific background band, and asterisks to the left of the gel mark NS4B-specific, PNGase F-sensitive bands. (C) The indicated 293T cells were cotransfected to express NS4B-HA and either NS1 or GFP (as transfection controls). Cell lysates were treated with PNGase F to facilitate quantitation of NS4B protein by removing N-glycans, and Western blotting was performed for the indicated proteins. Chemiluminescent band intensities were quantified as NS4B-HA in STT3B knockout cells relative to NS4B-HA in WT cells compared to the same ratio of GFP or NS1 to control for transfection efficiency. Each point plotted is the quantification of an independent transfection, where the open triangles compare NS4B levels with NS1 and the closed circles compare NS4B levels with GFP. The mean and SD plotted are for all points. (D) The indicated 293T cells were transiently transfected to express NS4B-HA and treated with cycloheximide at 24 h posttransfection to block further translation. Cells were lysed at the indicated times after cycloheximide treatment, and proteins were resolved by Western blotting. (Left) Representative blots. (Right) Decay curves plotted with data from three independent experiments. Values are means ± SD. KO, knockout.

Fig 3: TUSC3 associates with oligosaccharyltransferase subunit STT3B.(a) TUSC3 co-immunoprecipitates with STT3B in HEK293T cells. Lysates from HEK293T cells were immunoprecipitated with TUSC3 antibody or control IgG and detected with STT3B antibody on a western blot. IgG heavy chain (IgG HC) represents the loading control. Endogenous input STT3B and TUSC3 levels are shown. (b) HEK293T cells were transfected with TUSC3 or an empty control pcDNA 3.1 vector (V). Lysates from transfected cells were immunoprecipitated using STT3B antibody or control IgG and TUSC3 was detected using western blot. IgG light chain (IgG LC) serves as loading control. TUSC3 overexpression affects ER-Luciferase N-glycosylation in HEK293T cells. (c) HEK293T cells were transfected with wild type firefly luciferase (Luc) or luciferase fused to an endoplasmic reticulum targeting sequence (ER-Luc) and seeded in 96-well plates. Luminesence was measured 32–48 hours after transfection and normalized to the cell count. (d) HEK293T cells were co-transfected with ER-Luc and empty pcDNA 3.1 vector (V) or full length TUSC3. Luminesence was measured and normalized to the cell count. The luciferase experiments are representative of several biological replicates and were performed in triplicates (depicted as means and standard deviations). Electrophoretic gel shift of the fusion construct (ER-Luc) in contrast to wild type luciferase (Luc) is shown. TUSC3 overexpression does not have an effect on the molecular weight of the ER-Luciferase. (e) A panel of cell lines derived from human prostate cancers (DU145, PC3, LNCaP) and benign prostatic hyperplasia (BPH-1) were assessed for expression of TUSC3, PTEN and ENTPD5. Cropped blots are depicted for clarity reasons and full-length blots will be provided upon request.

Fig 4: SARS-CoV-2 induces N-glycosylation through NF-?B/STT3A axis. (a) Locus compare plot for the comparison between the STT3A eQTLs and the GWAS result, the COVID-19 A2 analysis for patients with severe respiratory symptoms via COVID-19 Host Genetics Initiative (COVID-19-HGI) project. The points are colored based on linkage disequilibrium (LD) bins. rs201008304, the indel variant in strong LD with the lead STT3A eQTL, is the top and most significant SNP in COVID-19 A2 analysis. (b) The eQTL plot for the association between STT3A variant types [A/A (n = 437), A/AT (n = 46), and AT/AT (n = 0)] expression and rs201008304 in cells-cultured fibroblasts was queried from the GTEx. (c) Forest plot shows the risk of rs201008304 in different COVID-19 phenotypes, originally from the summarized statistic results. (d) Representative data of immunohistochemistry staining of STT3A (red cells) and N protein in lung tissues of AAV-Ctrl and AAV-hACE2 mice infected by SARS-CoV2 at 5 dpi. Images were captured at 40x objective lens magnification. Scale bar, 100 µm. Three randomly selected STT3A/nuclei expression fields in lung tissues were exported from CaseViewer software (3DHistech) and quantified by the HistoQuant plugin in QuantCenter (3DHistech). Statistical method: t test, *p < 0.05, (n = 3). (e) Western blotting of S protein in Vero E6 cells after knockdown of STT3A or STT3B. Circle, g-spike; arrow, ng-spike. (f) ChIP analysis of enrichment of NF-?B in STT3A promoters after SARS-CoV-2 infection for 1 d. The enrichment levels were analyzed by RT-qPCR and shown as the percentage of input. Statistical method: two-way ANOVA, Tukey post hoc tests, ***p < 0.001 (n = 3 with mean ± SD shown).

Fig 5: Perturbation of N-linked glycosylation distinctly impacts A2234D Tg.A, schematic detailing the N-glycosylation and lectin-mediated folding pathway. Glycosylation is carried out by two distinct OST complexes containing STT3A or STT3B as catalytic subunits. Glucosidases then trim terminal glucose residues, lectin chaperones (CANX, CALR), and PDIA3 assist in folding, followed by further glucose trimming. Subsequently, glucosyltransferases (UGGT1, UGGT2) serve as quality control sensors to re-glucosylate improperly folded proteins for iterative chaperoning cycles. B–E, interaction changes of Tg mutants compared with WT with individual N-glycosylation quality control factors that were identified as high-confidence interactors of Tg. Error bars show SEM. Student’s parametric t test with adjusted p values (Storey q values) was used to determine significant changes. *q < 0.05, **q < 0.005, ***q < 0.0005. B, lectin chaperones and lectin-associated protein disulfide isomerase PDIA3. C, glucosyltransferases sensing misfolded proteins. D, glucosidases involved in glycan trimming. E, catalytic subunits of the OST complex STT3A responsible for cotranslational glycosylation and STT3B responsible for posttranslational glycosylation. F, comparison of WT Tg secretion in parental, STT3A, or STT3B KO HEK293T cells. WT Tg was transiently transfected into the respective cells, and newly synthesized proteins were metabolically labeled for 30 min with 35S and then chased with unlabeled media. 35S-labeled protein was quantified in the media, and lysate after 4 h % Tg secreted was calculated as Tgmedia,4h/(Tglysate,0h+Tgmedia,0h). Error bars represent SEM of three to four biological replicates. STT3A or STT3B KO do not significantly alter WT secretion. Student’s parametric t test was used to determine significant changes in Tg secretion. Representative autoradiograms are shown in supplemental Figure S5G. G, comparison of A2234D Tg degradation in parental, STT3A, or STT3B KO HEK293T cells. A2234D Tg was transiently transfected into the respective cells and subjected to the same 35S-pulse labeling scheme as in F. % Tg degraded was calculated as 1-(Tglysate,4h/Tglysate,0h). Error bars represent SEM of three to four biological replicates. Representative autoradiograms are shown in supplemental Figure S5H. Student’s parametric t test was used to determine significant (*p < 0.05) changes in Tg degradation. OST, oligosaccharyl transferase complex; Tg, thyroglobulin; UGGT1, UDP-glucose glycoprotein glucosyltansferase 1;UGGT2, UDP-glucose glycoprotein glucosyltansferase 2.