Fig. 5: Agonist-induced formation of a 4-aaRS, SPEAR-binding complex.

a Immunoblots of size-exclusion chromatographic fractions of agonist-treated Calu-3 cells. b NSAP1 was immunoprecipitated from agonist-stimulated Calu-3 extracts treated as in a. NSAP1-binding aaRSs were eluted and detected by immunoblot (top). From same Calu3 extracts, aaRSs were subjected to IP with antibodies targeting aaRSs, and bound proteins detected by immunoblot (bottom). c Immunoblots of size-exclusion chromatographic fractions of agonist-treated A549-hACE2 cells. d Size-exclusion chromatography fractions from (c) were subjected to RNA EMSA with [α-³²P]UTP-labeled SPEAR as probe (top). Free probe from same gel at lower exposure (bottom). e Supershift RNA EMSA assay of d fraction 16 from spike S1- and IFN-γ-treated cells with antibodies against MSC constituents; two different anti-RARS1 and anti-KARS1 antibodies were used (top). Free probe at lower exposure (bottom). Vehicle is 50% glycerol in PBS. Experiment in b was done once, and experiments in a, c, d, e were done three times. f Schematic of tetra-aminoacyl tRNA synthetase sarbecoviral RNA-interacting (TASRI) complex binding to SPEAR element. g ER and PNS isolated from agonist-stimulated A549-hACE2 cells were subjected to immunoblot with antibodies targeting MSC constituents and cell fraction markers. ER enrichment was determined as ER:PNS ratio by densitometry, and fold-change in agonist-stimulated cells was determined by normalizing with the ratio in untreated cells (n = 3 independent biological replicates; data are presented as mean values ± SD). h Stimulus-treated A549-hACE2 cells were fixed and subjected to confocal microscopic detection of the ER marker KDEL (green), DAPI (blue), and EPRS1 (magenta). Signal intensities were quantified using ImageProPlus, and ER localization of EPRS1 was quantified as fraction of total EPRS1 co-localizing with KDEL. Box represents 25th to 75th percentile and whiskers represent minimum to maximum values; central bars represent medians. (right, n = 27–44). p values from two-tailed Student’s t test. *Too low to calculate accurately.