Extended Data Fig. 7: K22 impairs the ability of NSP6 to organize the NSP3–NSP4 puncta and has anti-SARS-CoV-2 activity.
From: The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle
a, HeLa cells transfected with HA-NSP3 and mCherry-NSP4 for 5 h were further transfected or not with Flag-NSP6 and treated with DMSO or K22 (40 μM, 16 h) followed by immunostaining as indicated. b, Quantification of the number of NSP4 puncta/cell in (a). N = 3, n = 60. Single values are plotted. The median value is shown. One-way ANOVA test with Tukey’s post-hoc. ns, not significant. c, d, CLEM analysis of K22-treated cells. c, Fluoromicrograph of HeLa cell expressing HA-NSP3, mCherry-NSP4 and Myc-NSP6. Inset corresponds to the boxed area and shows NSP3/NSP4-positive structures (arrows) close to the NSP6 compartment (arrowhead). d, Overlap of fluorescent image (inset in c) with EM image. The NSP6 compartment corresponds to a circular zippered ER structure (arrowhead) close to but not connected with the NSP3/NSP4 puncta that correspond to DMVs (arrows). The empty arrow indicates a tubular connection of a DMV to the regular ER (magnified in the inset). e, Ultrastructure of DMV clusters in K22-treated cells expressing HA-NSP3, mCherry-NSP4 and Myc-NSP6. Serial sections show a DMV cluster with irregular elongated DMVs (black arrows). The empty arrow indicates a tubular connection of a DMV with regular ER. f, Morphometric analysis of serial sections from untreated (NT) and K22-treated cells to quantify the number of DMVs per cluster, DMV shape factor, and the number of tubular or zippered connections per DMV cluster. Single values are plotted, Medians are shown, n = at least 8 clusters or 70 DMVs, two-tailed unpaired t-test. g, h, Antiviral activity of K22. Effects of K22 on cell death measured by LDH (g) or on viral replication (h) in SARS-CoV-2-infected Calu-3 cells. Mean ± SEM, N = 3, one-way ANOVA. i, Calu-3 cells infected with SARS-CoV-2 without (left panels) or with (right panel) K22 treatment. Cells were immunostained for dsRNA and NSP6. Nuclei were stained with DAPI. An enlargement of the boxed area shows NSP6 labelling (arrowheads) in the proximity of replication areas labelled by dsRNA. Graph, NSP6 structures within a distance of 250 nm from dsRNA spots, and dsRNA spots within a distance of 250 nm from NSP6 structures were counted and expressed as a percentage of total. Mean ± SEM, 15 cells analysed, n = 729 NSP6 structures, n = 901 dsRNA spots. Scale bar, a, 10 μm; c, 4.4 µm; d, 370 nm; e, 320 nm; i, 20 μm.
