Fig 2: N-terminusdetermines the extra processing and mitochondriallocalization of NSP3A. Schematic illustration of chimeric plasmids used in this figure. B. The processing site of CoV2-NSP3 is localized at its N-terminus. Green arrow indicates the released EGFP. NOex indicates no exchange, PLproex indicates the exchange of PLpro domain, whereas Ubl1–HVRex indicates the exchange of Ubl1 and HVR domains. Asterisks indicate non-specific signals. C. The N-terminus of CoV2-NSP3 re-locates CoV1-NSP3 to mitochondria. Scale bar, 10 µm. CANX, Calnexin (an ER marker); ER, endoplasmic reticulum.

Fig 3: Wild-type SEC23B localizes to the nucleus.A Non-malignant thyroid Nthy-ori 3-1 cells were stably transduced with EGFP, wild-type SEC23B, and mutant p.Val594Gly SEC23B (the latter two fused with EGFP). Nthy-SEC23B-WT and Nthy-SEC23B-V594G cells show nuclear expression of SEC23B. Nthy-EGFP cell line is used as a control to show non-specific GFP subcellular localization. Blue, DAPI; green, SEC23B-EGFP or EGFP. Scale bars, 25 µm. B Protein levels after nuclear–cytoplasmic fractionation in stably transduced Nthy-ori 3-1 cells detects endogenous and EGFP-tagged SEC23B in the nuclear compartment. Nthy-EGFP cell line is used as a control to indicate baseline endogenous levels of SEC23B. PARP1, nuclear marker; GAPDH, cytoplasmic marker; CANX, endoplasmic reticulum marker. C Protein levels after nuclear–cytoplasmic fractionation in parental HEK293 cells detects endogenous SEC23B in the nuclear compartment. PARP1 and Lamin A/C, nuclear markers; GAPDH, cytoplasmic marker; CANX, endoplasmic reticulum marker.

Fig 4: CRT promotes the secretory trafficking of ATZ in Huh7.5 cells.A, representative flow cytometry dot plots of cellular eYFP fluorescence in untransfected, eYFP-AAT–transfected, or eYFP-ATZ–transfected Huh7.5 CRT-knockout (CRT KO) and WT (control vector) cells. B, total live cells expressed as a percentage of all cells (pre-gated on forward and side scatter), and percentage of eYFP+ cells identified from the total live cell population. C, total media fluorescence and cell MFI values were normalized relative to corresponding values from CRT KO cells in eYFP-AAT–transfected or eYFP-ATZ–transfected cells. D, the ratio between the media and cell fluorescence was obtained, as in Fig. 1. Data in (B–D) were obtained from 16 independent transfections of the Huh7.5 CRT KO and WT lines and are shown as mean ± S.D. Nine replicates were done in parallel with CNX data in Fig. 4. Paired two-tailed t tests were performed comparing CRT KO with WT conditions for each measurement. Because the data in panel C is normalized, for this panel, t tests were performed on log-transformed data. *p <0.05, ****p <0.0001. See also Fig. S3, D–F for individual experimental trends of the eYFP-ATZ data.

Fig 5: Chaperone proteins calnexin (CANX) and calreticulin (CALR) regulate SARS-CoV-2 viral entry.(A) Parent Spike was transfected into WT, CANX-KO, and CALR-KO 293Ts. Spike expression was similar in all cells based on anti-FLAG that detects the Spike C terminus (left). A partial (10 to 25%) reduction in anti-RBD Ab/Spike S1 association (middle) was noted, and this correlated with ACE2-Fc binding (right). (B) 293Ts were transfected with parent Spike. Spike immunoprecipitated from cell lysate 24 hours after transfection using anti-FLAG mAb and coprecipitated both CANX and CALR. CALR (top), CANX (middle), and Spike (bottom) were analyzed both in the immunoprecipitated (protein A/G “bead”) sample and the supernatant remaining after bead removal. Arrows and boxes indicate target proteins. (C) All Spike mutants coprecipitated CANX in the anti-FLAG Spike pulldown assay. Cells without Spike (“untransfected”) served as negative control. (D) Parent pseudotyped lentivirus was produced in WT, CANX-KO, and CALR-KO. Western blot showed reduced Spike incorporation into virions produced in both KOs. (E) Pseudovirus entry into 293T/ACE2, measured 72 hours after infection, was reduced when virus were produced in CANX or CALR-KO 293Ts. (F) VSV-G pseudotyped lentivirus entry into 293T/ACE2 was not reduced by either CANX or CALR. (G) Luc-VLP containing parent Spike were produced in WT, CANX-KO, and CALR-KO 293Ts. Luc-VLPs produced in CANX-KO displayed reduced entry into A549-ACE2-TMPRSS2, Calu-3, and 293T/ACE2 cells. Luc-VLP viral entry is reported on the basis of luminescence signal, normalized to parent levels in each run. No spike Luc-VLP lacks Spike. Data are means ± SD (n = 3). *P < 0.05 with respect to all other treatments.