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. 2021 Oct 11;56(23):3250–3263.e5. doi: 10.1016/j.devcel.2021.10.006

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© 2021 Elsevier Inc.

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Ser171 and Trp193 are essential for ORF3a to promote lysosomal exocytosis and to block autophagy

(A) Schematic illustration of the domains in ORF3a of SARS-CoV-2 (SARS2) and SARS-CoV (SARS).

(B) Quantification of the number of VPS39-GFP puncta in cells expressing ORF3a-mCherry, ORF3a(42aa–275aa)-mCherry, ORF3a(S171E)-mCherry, SARS ORF3a-mCherry, SARS ORF3a(42aa–274aa)-mCherry, and ORF3a(W193R)-mCherry. Data are shown as mean ± SEM (n = 31, 22, 32, 32, 32, and 25 for bars from left to right). ^∗∗∗p < 0.001; n.s., no significant difference.

(C and D) Expression of SARS-CoV-2 ORF3a (D) but not SARS-CoV ORF3a (C) induces the formation of a large number of VPS39 puncta.

(E) Quantification of the number of VPS39-GFP puncta in cells expressing ORF3a-FLAG and a series of ORF3a-FLAG mutants with the indicated regions replaced with the corresponding regions of SARS-CoV ORF3a. Data are shown as mean ± SEM (n = 29 for ORF3a(165–171) chimeric mutants, n = 20 for other bars). ^∗∗∗p < 0.001; ^∗∗p < 0.01; n.s., no significant difference.

(F) The mutant ORF3a(S171E), in which Ser171 of SARS-CoV-2 ORF3a is replaced by Glu171 of SARS-CoV ORF3a, fails to induce formation of VPS39-GFP puncta.

(G and H) Compared with control cells expressing GFP (Figure 1C), the level of cell-membrane-localized LAMP1 detected by the live cell staining assay is not increased in cells expressing SARS-CoV ORF3a-GFP (G) or SARS-CoV-2 ORF3a(S171E)-GFP (H).

(I) Quantification of the fluorescence intensity of cell-membrane-localized LAMP1 per cell. The level in control cells is set to 1.0. Data are shown as mean ± SEM (n = 33, 23, 21, 28, 29, 23, and 16 for bars from left to right). ^∗p < 0.05, ^∗∗∗p < 0.001; n.s., no significant difference. The data for control and ORF3a-expressing cells are the same as those in Figure 1E.

(J) Quantification of the number of STX4-GFP and VAMP7-GFP puncta in cells expressing mCherry (control), ORF3a-mCherry, ORF3a(S171E)-mCherry, and SARS-CoV ORF3a-mCherry. Data are shown as mean ± SEM (n = 16, 30, 24, 19, 14, 31, 22, and 19 for bars from left to right). ^∗∗∗p < 0.001. The data for ORF3a-expressing cells are the same as those in Figure 3C.

(K) In GFP-Trap assays, FLAG-VPS39 is co-precipitated by ORF3a-GFP and very weakly by ORF3a(W193R)-GFP, but not by ORF3a(S171E)-GFP. Cell lysates were immunoprecipitated using GFP-Trap and analyzed by immunoblotting with FLAG and GFP antibodies.

Scale bars: (C, D, F, G, and H) 10 μm.

See also Figures S5 and S6.