Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: SARS-CoV-2 Mpro cleaves MAGED2 at Gln-263. ( A ) Putative Mpro cleavage sites in SARS-CoV-2 non-structural proteins and MAGED2. ( B and C ) HEK293T cells were co-transfected with human MAGED2 or Q263N mutant with Flag tag at C-terminal and HA-tagged SARS-CoV-2 Mpro or a proteolytically inactive mutant Mpro (C145A). Lysates from transfected cells were prepared for immunoblotting with antibodies, as indicated. ( D ) MAGED2 mutant with Flag tag at C-terminal and HA-tagged Mpro were co-expressed in HEK293T cells. Lysates from transfected cells were prepared for immunoblotting with antibodies, as indicated. ( E ) MAGED2 cleavage assay in vitro . Purified MAGED2 and Mpro wild-type (WT) or C145A mutant proteins were incubated in vitro and analyzed by Coomassie blue staining. One star is MAGED2 N , and two stars indicate MAGED2 C . ( F ) A549-hACE2 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 or 1, and immunoblot was performed at 24-h post-infection. Red star indicates cleaved MAGED2. Each experiment was independently repeated three times with similar results, and the representative images are shown.

Article Snippet: SARS-CoV-2 proteins, MAGED2 full-length, and its truncation expressing plasmids were constructed into pLVX-IRES-zsGreen1 by 2× MultiF Seamless Assembly Mix (RK21020, Abclonal, China).

Techniques: Transfection, Mutagenesis, FLAG-tag, Western Blot, Cleavage Assay, In Vitro, Purification, Incubation, Staining, Infection

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: MAGED2 cleavage by Mpro is conserved in multiple mammalian species and coronaviruses. ( A ) A phylogenetic tree was constructed based on the protein sequences of MAGED2 orthologs by using the neighbor-joining method conducted in program MEGA6. MAGED2 residues neighboring Mpro cleavage site from human, rhesus macaque, White-tufted-ear marmoset, tufted capuchin, Sunda flying lemur, Brandt’s bat, goat, cattle, horse, Malayan pangolin, dog, domestic ferret, domestic cat, and house mouse are aligned. ( B to E ) HEK293T cells were transfected with MAGED2 orthologs as indicated species. The uncleaved or cleaved protein band intensity was quantitatively analyzed using ImageJ. Cleavage efficiency = cleaved products/(cleaved products + uncleaved protein) × 100% ( B ), MAGED2 mutant S264P ( C ), and HA-tagged Mpro from SARS-CoV-2, other coronavirus (SARS-CoV or MERS-CoV) ( D ), or SARS-CoV-2 variants (Beta or Omicron) ( E ). Lysates of transfected cells were analyzed by immunoblotting with the antibodies indicated on the left. Western blots are quantified with ImageJ. Each experiment was independently repeated three times with similar results, and the representative images are shown. Values are means plus standard deviations (error bars) from one representative experiment with three biological replicate samples. **, P < 0.01 by one-way analysis of variance.

Article Snippet: SARS-CoV-2 proteins, MAGED2 full-length, and its truncation expressing plasmids were constructed into pLVX-IRES-zsGreen1 by 2× MultiF Seamless Assembly Mix (RK21020, Abclonal, China).

Techniques: Construct, Transfection, Mutagenesis, Western Blot

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: MAGED2 is a restriction factor that inhibits SARS-CoV-2 infection. ( A ) Caco-2-N cells were transduced with sgRNA targeting MAGED2. Whole-cell lysate was analyzed by immunoblotting assay at 5-day post-transduction. ( B and C ) WT or MAGED2-depleted Caco-2-N cells were infected with SARS-CoV-2 GFP/ΔN trVLP at an MOI of 0.1. After 24 h, cells were analyzed by flow cytometry to determine the percentage of SARS-CoV-2 GFP/ΔN trVLP-infected cells. Data are normalized with non-targeting control ( B ). Meanwhile, intracellular RNAs were purified for RT-qPCR assay to quantify SARS-CoV-2 genomic RNAs ( C ). ( D ) Human MAGED2 was 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. ( E and F ) MAGED2 knockout Caco-2 cells were infected with SARS-CoV-2 authentic virus at an MOI of 0.1. After 24 h, viral particles in the supernatant were titrated ( E ), and intracellular RNAs were purified for RT-qPCR assay to quantify SARS-CoV-2 subgenomic E RNAs ( F ). Values are means + standard deviations (error bars) from one representative experiment with three biological replicate samples, and each experiment was repeated three times. **, P < 0.01; ***, P < 0.001 by one-way analysis of variance.

Article Snippet: SARS-CoV-2 proteins, MAGED2 full-length, and its truncation expressing plasmids were constructed into pLVX-IRES-zsGreen1 by 2× MultiF Seamless Assembly Mix (RK21020, Abclonal, China).

Techniques: Infection, Transduction, Western Blot, Flow Cytometry, Purification, Quantitative RT-PCR, Knock-Out, Virus

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: MAGED2 inhibits SARS-CoV-2 genome replication but not restricts viral entry, assembly, and release. ( A and B ) HeLa-ACE2 cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2, ACE2, and β-actin antibodies. The HeLa-ACE2 cells with or without MAGED2 depletion were infected with MLV retroviral particles (Fluc as the reporter) pseduotyped with SARS-CoV-2 spike (MLV SARS-CoV-2pp). Fluc activity was measured at 48-h post-infection. 1F11 is SARS-CoV-2 neutralizing antibody as the positive control. ( C ) Schematic representation of SARS-CoV-2 Gluc replicon RNA genome. ( D and E ) Caco-2-N cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-Actin antibodies. Then, the cells with or without MAGED2 genetic ablation were transfected with SARS-CoV-2 Gluc WT or SAA (RdRp inactive mutant) replicon RNAs, and Gluc activity was assayed at 48-h post-transfection. ( F ) Schematic representation of VLP production and detection. ( G and H ) HEK293T cells were transduced with sgRNA lentivirus targeting MAGED2. Whole-cell lysates were analyzed by immunoblot with MAGED2 and β-actin antibodies. Then, the HEK293T cells with or without MAGED2 knockout in 10 cm dish were transfected with equal amounts of plasmids (24 µg in total) encoding the SARS-CoV-2 S, E, M, and HiBiT-N proteins. After 24 h, cell culture supernatants were collected. VLPs separated by 10%–60% sucrose gradient centrifugation were measured with Nano-Glo luciferase kit. All data are representative of three independent experiments. Values are means + standard deviations (error bars) ( n = 3). *, P < 0.05; **, P < 0.01; n.s., not significantly different by one-way analysis of variance.

Article Snippet: SARS-CoV-2 proteins, MAGED2 full-length, and its truncation expressing plasmids were constructed into pLVX-IRES-zsGreen1 by 2× MultiF Seamless Assembly Mix (RK21020, Abclonal, China).

Techniques: Transduction, Western Blot, Infection, Activity Assay, Positive Control, Transfection, Mutagenesis, Knock-Out, Cell Culture, Gradient Centrifugation, Luciferase

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: 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, MAGED2 N (1-263 aa)-EGFP or MAGED2 C (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, MAGED2 N , MAGED2 C , or MAGED2 N (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 MAGED2 N (1-263 aa) or MAGED2 N (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.

Article Snippet: SARS-CoV-2 proteins, MAGED2 full-length, and its truncation expressing plasmids were constructed into pLVX-IRES-zsGreen1 by 2× MultiF Seamless Assembly Mix (RK21020, Abclonal, China).

Techniques: Infection, Virus, Immunoprecipitation, Western Blot, Transfection, Magnetic Beads, Electroporation, Quantitative RT-PCR, Transduction, Staining, Flow Cytometry

Journal: mBio

Article Title: SARS-CoV-2 main protease cleaves MAGED2 to antagonize host antiviral defense

doi: 10.1128/mbio.01373-23

Figure Lengend Snippet: Mpro cleaves MAGED2 to antagonize its antiviral activity. Model depicts that MAGED2 restricts SARS-CoV-2 replication by decreasing the interaction between N protein and viral genome through its N-terminal region. Mpro cleaves MAGED2 at Gln-263, and MAGED2 N translocated into the nucleus, which relieving its antiviral effect.

Article Snippet: SARS-CoV-2 proteins, MAGED2 full-length, and its truncation expressing plasmids were constructed into pLVX-IRES-zsGreen1 by 2× MultiF Seamless Assembly Mix (RK21020, Abclonal, China).

Techniques: Activity Assay