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. 2023 Jul 13;14(4):e01373-23. doi: 10.1128/mbio.01373-23

Fig 5.

Fig 5

MAGED2 interacts with viral N protein to disturb the association of N with viral RNA genome. (A) Caco-2 cells were infected with SARS-CoV-2 authentic virus at an MOI of 0.1. After 24 h, cells were collected and lysed. Cell lysates were immunoprecipitated with MAGED2 antibody or IgG control with/without 50 µg/mL RNase A treatment. Immunoprecipitants were subjected for immunoblotting assay with MAGED2 and N antibodies. (B) HA-N protein and Flag tagged MAGED2, MAGED2N (1-263 aa)-EGFP or MAGED2C (264-606 aa) were transfected into HEK293T cells. After 48 h, cell lysates were immunoprecipitated by Flag antibody-conjugated magnetic beads. Immunoprecipitants were subjected for immunoblotting assay with Flag and HA antibodies. (C and D) Schematic representation of RNA immunoprecipitation (RIP) assay. SARS-CoV-2 Gluc replicon RNAs, plasmids encoding GFP-Flag or N-Flag and HA-MAGED2 full-length or C-terminal truncation were co-electroporated into HEK293T cells. RIP was performed at 24-h post-electroporation as indicated, and RT-qPCR assay was conducted to determine the RNA abundances. The precipitated RNA was normalized with input. (E) Subcellular localization of MAGED2 full-length and its truncations. Flag-tagged MAGED2 full-length, MAGED2N, MAGED2C, or MAGED2N(ΔNLS) was expressed in Caco-2 cells by lentiviral transduction. Cells were stained with Flag antibody, and the nuclei were stained with DAPI. (F) HA-tagged N protein and Flag-tagged MAGED2N (1-263 aa) or MAGED2N (ΔNLS) were transfected into HEK293T cells. After 48 h, cell lysates were immunoprecipitated by Flag antibody-conjugated magnetic beads. Immunoprecipitants were subjected for immunoblotting assay with Flag and HA antibodies. (G) Human MAGED2 full-length or its truncations were ectopically expressed in Caco-2-N cells by lentiviral transduction, and the cells were subsequently infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. Cells were analyzed by flow cytometry at 24-h post-infection to determine the percentage of the trVLP-infected cells. Values are means + standard deviations (error bars) (n = 3). ***, P < 0.001; n.s., not significantly different by one-way analysis of variance.