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. 2021 Apr 13;12(2):e00065-21. doi: 10.1128/mBio.00065-21

FIG 3.

FIG 3

ORF6 of SARS-CoV-2 results in reduced mCherry reporter protein expression in 293T cells. (A) Schematic representation of ORF6 constructs used in this study. (B) 293T cells were transiently transfected with GFP-tagged constructs and mCherry and visualized 48 h after transfection. All images were taken with identical fluorescence gain settings. Bars, 100 μm. (C) The fluorescent intensities for three fields per cotransfection were measured with ImageJ and displayed as means ± standard errors (error bars). Wild-type (WT) ORF6 caused a significant reduction in mCherry expression. ORF6 constructs with deletions or a single amino acid substitution (Met58Ala) in the C terminus showed a smaller reduction in mCherry expression than WT ORF6. Cell lysates were collected for each of the conditions. (D) Western blotting confirmed the role of the C terminus of ORF6 in reducing protein expression in transfected cells. (E) Poly(A) mRNA staining of transiently transfected 293T cells revealed ORF6 Met58Ala-expressing cells do not accumulate mRNA in the nuclei, consistent with the results from the mCherry cotransfection assay. Bars, 5 μm. *, P < 0.05; **, P < 0.01.