Extended Data Fig. 2: Analysis of the ability of ACE2 orthologs to support the entry of MRCoV and analysis of expression of NvACE2 mutants.

a, HeLa cells were transfected with expression plasmids for Flag-tagged ACE2 from mink, Pipistrellus abramus (P.abramus), human, pangolin, Eurasian badger, monkey, pig, raccoon dog, and camel or with empty expression vector and infected with MRCoV (m.o.i. = 1). At 48 h.p.i. cells were fixed and stained for ACE2 expression and for dsRNA (a replication intermediate of the virus), nuclei were counterstained with DAPI. Red, ACE2; green, dsRNA; blue, nuclei; scale bar, 10 μm. The representative images from three independent experiments are shown. b, Replication dynamics of MRCoV in HeLa cells expressing ACE2 proteins from indicated species. Viral RNA copies in the supernatants were determined by RT-qPCR. Data are presented as the mean ± s.d. of three independent experiments. Statistical analysis was performed using one-way ANOVA. c, HEK 293 T cell stably expressing WT NvACE2 and its mutants with indicated substitutions were fixed and subjected to IFA using anti-Flag antibodies; nuclei were counter-stained with DAPI. Green, NvACE2 and its variants; blue, nuclei; scale bar, 500 μm. The representative images from three independent experiments are shown. Percentages of NvACE2 positive cells relative to DAPI positive nuclei were calculated by Image J. Data are presented as the mean ± s.d. of measurements performed for three randomly selected fields.