Extended Data Fig. 1: NSP6 requires a free C terminus to exert its membrane-deforming activity.
From: The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle
a, Fluoromicrographs of Calu-3 cells expressing YFP-Cb5 alone (leftmost panels) or in combination with C-terminally or N-terminally Flag-tagged NSP6, or untagged NSP6 (NSP6), as indicated, and immunostained with anti-Flag antibody (red). b, Expression analysis of NSP6-Flag (C-term) or Flag-NSP6 (N-term) in transfected HeLa cells. Left, representative fluorescence micrographs (anti-Flag antibody). Right, fluorescence intensity measurements. Single values are plotted, Means ± SEM are indicated. N = 3, n = 69 cells. ns, not significant. Two-tailed unpaired t-test with Welch’s correction. Lower panel, western blot of total protein lysates using an anti-Flag antibody; actin was used as a loading control. NT, non-transfected cells. c, Western blot of HeLa cells expressing HA-NSP6 or untagged NSP6, detected using anti-HA or anti-NSP6 antibody, as indicated. NT, non-transfected cells. d, HeLa cells transfected with Flag-NSP6 immunostained with anti-Flag, anti-LAMP1, anti-EEA1 or anti-LC3 antibodies. e, Fluoromicrographs of HeLa cells expressing YFP-Cb5 and either IBV NSP6-Flag (upper panels) or IBV-Flag-NSP6 (lower panels). Cells immunostained with anti-Flag antibody (red). f, IEM (anti-HA immuno-gold-labelling) of a HeLa cell expressing HA-NSP6. g, Magnification of the boxed area. Arrows show the regular single membrane of the ER cisterna; arrowheads indicate zippered membranes in the circular NSP6-positive structure. h, Single slices from a tomogram of a HeLa cell expressing HA-NSP6. Connections of circular zippered structures with the ER are shown by black arrows, and the connection of linear zippered membranes with the nuclear envelope is indicated by a white arrow (see Supplementary Video 1). i, Routine EM of a HeLa cell expressing HA-NSP6 and j, magnification of boxed area. The arrow indicates apposition of limiting membranes of an ER cisterna (asterisk) that then continues into the zippered ER domain (arrowheads). Western blots in b,c are representative of three independent experiments each. Scale bars, a, b, d, e, 10 µm; f, g, 100 nm; h, 230 nm; i, 200 nm; j, 100 nm.
