Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: CRISPR activation screen identifies MFGE8 as a proviral host factor for rVSV-CCHFV infection. ( A ) Identification of genes from CRISPR screen in A549-BAT cells. Cells transduced with the CRISPR activation library were infected with rVSV-CCHFV for 24 h. GFP-positive cells were sorted for sgRNA abundance analysis and ranked based on the MAGeCK score and P value. ( B and C ) Validation of MFGE8 gene. Gene expression was activated using two or representative sgRNAs in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 18 h) ( B ) and rVSV (MOI 0.01, 15 h) ( C ). The percentage of GFP-positive cells were analyzed by flow cytometry. ( D ) Representative fluorescence images of A549-BAT cell infected with respective virus from ( B ) and ( C ) were taken before harvesting the cells. Scale bar, 400 µm. ( E ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-BAT cells. ( F ) Growth kinetics of rVSV-CCHFV in vector control and MFGE8-overexpressing cells. Cells were infected with rVSV-CCHFV at an MOI of 0.3, and viral titers in the supernatants at indicated time points were determined by plaque-forming assay. ( G–I ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-WT ( G ), Hela ( H ), and SW-13 ( I ) cells. The percentage of GFP-positive cells were analyzed by flow cytometry at 16 hpi. (J) Knockout of MFGE8 decreases the rVSV-CCHFV infection. A549-WT cells edited with two different nontargeting control or MFGE8 -specific sgRNAs were infected with rVSV-CCHFV, followed by flow cytometry analysis of GFP-positive cells at 16 hpi. Two-way ANOVA with Sidak’s multiple-comparison test. ns, not significant; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human recombinant MFGE8 protein with His tag at the C-terminus was purchased from SinoBiological (10853-H08B).

Techniques: CRISPR, Activation Assay, Infection, Transduction, Biomarker Discovery, Gene Expression, Flow Cytometry, Fluorescence, Virus, Over Expression, Plasmid Preparation, Control, Knock-Out, Comparison

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: MFGE8 promotes the infection of pseudotyped, tecVLP, and authentic CCHFV across multiple strains. ( A and B ) Overexpression of MFGE8 in A549-BAT ( A ) and SW-13 ( B ) cells promotes the infection of pseudotyped CCHFV across multiple strains. The single-round VSV-based pseudovirus particles were packaged using the GPCs from different strains (Oman, Turkey, Afg2990, YL16070). The luciferase activity was determined at 24 hpi. ( C ). Knockout of MFGE8 decreases the infection of pseudotyped CCHFV across multiple strains. A549-WT cells edited with control or MFGE8-specific sgRNA were infected with pseudoviruses, and the luciferase activity was determined at 24 hpi. ( D and E ) Overexpression of MFGE8 enhances authentic CCHFV infection in A549-BAT ( D ) and A549-WT ( E ) cells. Cells were infected with CCHFV (YL16070 strain) at indicated MOIs for 72 h, and total cellular RNA were extracted for RT-qPCR. Relative viral RNA levels of S genome were analyzed by normalizing to internal GAPDH mRNA and then to the vector control. ( F ) Knockout of MFGE8 decreases authentic CCHFV infection. A549-WT cells edited with control or MFGE8 -specific sgRNA were infected with CCHFV (YL16070 strain) at indicated MOIs for 72 h. RT-qPCR analysis was performed as did for the overexpressing cells above. ( G and H ) A549-BAT cells overexpressing MFGE8 were infected with either Oman ( G ) or Turkey ( H ) tecVLPs, and NanoLuc luciferase activity was measured at 24 hpi. ( I ) MFGE8 -knockout A549 cells were infected with Turkey tecVLP, and NanoLuc activity was measured at 24 hpi. Unpaired t -tests. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Human recombinant MFGE8 protein with His tag at the C-terminus was purchased from SinoBiological (10853-H08B).

Techniques: Infection, Over Expression, Luciferase, Activity Assay, Knock-Out, Control, Quantitative RT-PCR, Plasmid Preparation

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: MFGE8 mediates rVSV-CCHFV entry into host cells through integrin. ( A-B ). Virus binding and internalization assays in overexpressing ( A ) or gene-edited ( B ) cells. Cells were incubated with rVSV-CCHFV (MOI 5), and the bound or internalized virions were measured by RT-qPCR for genomic RNA. The relative amount of bound or internalized virions was normalized to internal control GAPDH, and then to the control cells. ( C ). Cells pre-incubated with MFGE8 protein facilitate rVSV-CCHFV infection. A549-BAT cells were pre-incubated with different concentrations of MFGE8 protein at 37°C for 4 h, then infect with rVSV-CCHFV (MOI 3, 18 h) or rVSV (MOI 0.01, 15 h). The percentage of GFP-positive cells was analyzed by flow cytometry. ( D ). rVSV-CCHFV particles pre-incubated with MFGE8 protein facilitate virus infection. The rVSV-CCHFV particles (MOI 3) were pre-incubated with different concentrations of MFGE8 protein or mpox virus B6 control protein at 37°C for 1 h, then inoculated the A549-BAT cells for 18 h. The percentage of GFP-positive cells were analyzed by flow cytometry. ( E and F ) TecVLPs pre-incubated with MFGE8 protein facilitates virus infection. TecVLPs of IbAr10200 or Turkey stain were pre-incubated with varying concentrations of MFGE8 protein at 37°C for 1 h, then used to infect A549-BAT cells pre-transfected with plasmids expressing the L and N proteins. After 15 h of infection, NanoLuc activity was quantified. ( G ). Overexpression of MFGE8 with D48E mutation decreases the rVSV-CCHFV (MOI 3, 20 h) infection in A549-BAT cells. The percentage of GFP-positive cells were analyzed by flow cytometry. ( H-I ). The inhibitor of integrins decreases the rVSV-CCHFV infection. The MFGE8-overexpressing A549-BAT cells were pre-treated with integrin inhibitor cilengitide for 2 h at 37°C, and then infected with rVSV-CCHFV (MOI 3, 20 h) for flow cytometry analysis ( H ). The representative images were taken with fluorescence microscope ( I ). Scale bar, 400 µm. ( J ). MFGE8-overexpressing A549-BAT cells were edited with control, ITGB5-, or ITGB3-specific sgRNAs, and infected with rVSV-CCHFV (MOI 3, 17 h), followed by flow cytometry analysis. ( K ). A549-WT cells edited with control or ITGB5 sgRNA, followed by infection with rVSV-CCHFV (MOI 0.5, 16 h) for flow cytometry analysis. Unpaired t -tests ( A, B, K ), One-way ANOVA with Dunnett’s multiple comparisons test ( E, F, G, H, J ). ns, not significant; *, P < 0.1, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Human recombinant MFGE8 protein with His tag at the C-terminus was purchased from SinoBiological (10853-H08B).

Techniques: Virus, Binding Assay, Incubation, Quantitative RT-PCR, Control, Infection, Flow Cytometry, Staining, Transfection, Expressing, Activity Assay, Over Expression, Mutagenesis, Fluorescence, Microscopy

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: MFGE8 directly binds to the Gc domain of CCHFV. ( A ) The structure of MFGE8 protein predicted by AlphaFold2. Three domains, EGF, C1, C2, are indicated. ( B ) Schematic of different truncations of MFGE8 protein. ( C ) Overexpression of different truncated forms of MFGE8 in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 20 h) and flow cytometry analysis. ( D ) Schematic of chimeric MFGE8 proteins. The full-length MFGE8 without signal peptide was fused with the type II transmembrane protein, TMPRSS2. The C1 and C2 domains of MFGE8 were fused with type I transmembrane protein, MXRA8. The extracellular region of TMPRSS13 or MXRA8 was replaced. ( E ). Overexpression of chimeric MFGE8 proteins in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 20 h) and flow cytometry analysis. ( F ) Overexpression of different mutants of MFGE8 in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 20 h) and flow cytometry analysis. One-way ANOVA with Dunnett’s multiple comparisons test (C, E, and F). ns, not significant; * P < 0.1; *** P < 0.001; **** P < 0.0001. ( G ) Binding of MFGE8 protein to CCHFV Gc monomer, Gn, and Mpox virus B6 control protein. These proteins were pre-coated onto ELISA plates at a concentration of 10 µg/mL, followed by incubation of different concentrations of MFGE8 protein. The binding was evaluated using anti-MFGE8 and HRP-conjugated secondary antibodies. ( H–K ) MFGE8 and B6 control proteins were coated onto ELISA plates at a concentration of 10 µg/mL. Then, different concentrations of CCHFV Gc monomer ( H ), Gc trimer ( I ), or rVSV-CCHFV particles ( J ) were incubated and detected using anti-CCHFV-Gc ADI-36121 antibody and HRP-conjugated secondary antibody. ( K ) The binding affinity of MFGE8 to CCHFV Gc domain. His-tagged MFGE8 protein was immobilized onto the Ni-NTA biosensors. Binding parameters of recombinant Gc monomer to MFGE8 protein were measured by biolayer interferometry (BLI). Using a 1/1 binding model to derive equilibrium dissociation constant (Kd) values. ( L ) The model of soluble MFGE8-mediated cell entry of CCHFV. The soluble MFGE8 protein binds directly to both Gc protein and phosphatidylserine (PtdSer) on the surface of CCHFV virions and acts as a bridge by binding to the integrins on the cell membrane to promote virus infection.

Article Snippet: Human recombinant MFGE8 protein with His tag at the C-terminus was purchased from SinoBiological (10853-H08B).

Techniques: Over Expression, Infection, Flow Cytometry, Binding Assay, Virus, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Recombinant, Membrane

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: CRISPR activation screen identifies MFGE8 as a proviral host factor for rVSV-CCHFV infection. ( A ) Identification of genes from CRISPR screen in A549-BAT cells. Cells transduced with the CRISPR activation library were infected with rVSV-CCHFV for 24 h. GFP-positive cells were sorted for sgRNA abundance analysis and ranked based on the MAGeCK score and P value. ( B and C ) Validation of MFGE8 gene. Gene expression was activated using two or representative sgRNAs in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 18 h) ( B ) and rVSV (MOI 0.01, 15 h) ( C ). The percentage of GFP-positive cells were analyzed by flow cytometry. ( D ) Representative fluorescence images of A549-BAT cell infected with respective virus from ( B ) and ( C ) were taken before harvesting the cells. Scale bar, 400 µm. ( E ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-BAT cells. ( F ) Growth kinetics of rVSV-CCHFV in vector control and MFGE8-overexpressing cells. Cells were infected with rVSV-CCHFV at an MOI of 0.3, and viral titers in the supernatants at indicated time points were determined by plaque-forming assay. ( G–I ) Overexpression of MFGE8 enhances rVSV-CCHFV infection in A549-WT ( G ), Hela ( H ), and SW-13 ( I ) cells. The percentage of GFP-positive cells were analyzed by flow cytometry at 16 hpi. (J) Knockout of MFGE8 decreases the rVSV-CCHFV infection. A549-WT cells edited with two different nontargeting control or MFGE8 -specific sgRNAs were infected with rVSV-CCHFV, followed by flow cytometry analysis of GFP-positive cells at 16 hpi. Two-way ANOVA with Sidak’s multiple-comparison test. ns, not significant; *** P < 0.001; **** P < 0.0001.

Article Snippet: To construct the overexpressing cells, human MFGE8 cDNA was purchased from SinoBiological ( NM_005928.1 ) and cloned into pLV-EF1α-IRES-Puro vector (Addgene #85132).

Techniques: CRISPR, Activation Assay, Infection, Transduction, Biomarker Discovery, Gene Expression, Flow Cytometry, Fluorescence, Virus, Over Expression, Plasmid Preparation, Control, Knock-Out, Comparison

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: MFGE8 promotes the infection of pseudotyped, tecVLP, and authentic CCHFV across multiple strains. ( A and B ) Overexpression of MFGE8 in A549-BAT ( A ) and SW-13 ( B ) cells promotes the infection of pseudotyped CCHFV across multiple strains. The single-round VSV-based pseudovirus particles were packaged using the GPCs from different strains (Oman, Turkey, Afg2990, YL16070). The luciferase activity was determined at 24 hpi. ( C ). Knockout of MFGE8 decreases the infection of pseudotyped CCHFV across multiple strains. A549-WT cells edited with control or MFGE8-specific sgRNA were infected with pseudoviruses, and the luciferase activity was determined at 24 hpi. ( D and E ) Overexpression of MFGE8 enhances authentic CCHFV infection in A549-BAT ( D ) and A549-WT ( E ) cells. Cells were infected with CCHFV (YL16070 strain) at indicated MOIs for 72 h, and total cellular RNA were extracted for RT-qPCR. Relative viral RNA levels of S genome were analyzed by normalizing to internal GAPDH mRNA and then to the vector control. ( F ) Knockout of MFGE8 decreases authentic CCHFV infection. A549-WT cells edited with control or MFGE8 -specific sgRNA were infected with CCHFV (YL16070 strain) at indicated MOIs for 72 h. RT-qPCR analysis was performed as did for the overexpressing cells above. ( G and H ) A549-BAT cells overexpressing MFGE8 were infected with either Oman ( G ) or Turkey ( H ) tecVLPs, and NanoLuc luciferase activity was measured at 24 hpi. ( I ) MFGE8 -knockout A549 cells were infected with Turkey tecVLP, and NanoLuc activity was measured at 24 hpi. Unpaired t -tests. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: To construct the overexpressing cells, human MFGE8 cDNA was purchased from SinoBiological ( NM_005928.1 ) and cloned into pLV-EF1α-IRES-Puro vector (Addgene #85132).

Techniques: Infection, Over Expression, Luciferase, Activity Assay, Knock-Out, Control, Quantitative RT-PCR, Plasmid Preparation

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: MFGE8 mediates rVSV-CCHFV entry into host cells through integrin. ( A-B ). Virus binding and internalization assays in overexpressing ( A ) or gene-edited ( B ) cells. Cells were incubated with rVSV-CCHFV (MOI 5), and the bound or internalized virions were measured by RT-qPCR for genomic RNA. The relative amount of bound or internalized virions was normalized to internal control GAPDH, and then to the control cells. ( C ). Cells pre-incubated with MFGE8 protein facilitate rVSV-CCHFV infection. A549-BAT cells were pre-incubated with different concentrations of MFGE8 protein at 37°C for 4 h, then infect with rVSV-CCHFV (MOI 3, 18 h) or rVSV (MOI 0.01, 15 h). The percentage of GFP-positive cells was analyzed by flow cytometry. ( D ). rVSV-CCHFV particles pre-incubated with MFGE8 protein facilitate virus infection. The rVSV-CCHFV particles (MOI 3) were pre-incubated with different concentrations of MFGE8 protein or mpox virus B6 control protein at 37°C for 1 h, then inoculated the A549-BAT cells for 18 h. The percentage of GFP-positive cells were analyzed by flow cytometry. ( E and F ) TecVLPs pre-incubated with MFGE8 protein facilitates virus infection. TecVLPs of IbAr10200 or Turkey stain were pre-incubated with varying concentrations of MFGE8 protein at 37°C for 1 h, then used to infect A549-BAT cells pre-transfected with plasmids expressing the L and N proteins. After 15 h of infection, NanoLuc activity was quantified. ( G ). Overexpression of MFGE8 with D48E mutation decreases the rVSV-CCHFV (MOI 3, 20 h) infection in A549-BAT cells. The percentage of GFP-positive cells were analyzed by flow cytometry. ( H-I ). The inhibitor of integrins decreases the rVSV-CCHFV infection. The MFGE8-overexpressing A549-BAT cells were pre-treated with integrin inhibitor cilengitide for 2 h at 37°C, and then infected with rVSV-CCHFV (MOI 3, 20 h) for flow cytometry analysis ( H ). The representative images were taken with fluorescence microscope ( I ). Scale bar, 400 µm. ( J ). MFGE8-overexpressing A549-BAT cells were edited with control, ITGB5-, or ITGB3-specific sgRNAs, and infected with rVSV-CCHFV (MOI 3, 17 h), followed by flow cytometry analysis. ( K ). A549-WT cells edited with control or ITGB5 sgRNA, followed by infection with rVSV-CCHFV (MOI 0.5, 16 h) for flow cytometry analysis. Unpaired t -tests ( A, B, K ), One-way ANOVA with Dunnett’s multiple comparisons test ( E, F, G, H, J ). ns, not significant; *, P < 0.1, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: To construct the overexpressing cells, human MFGE8 cDNA was purchased from SinoBiological ( NM_005928.1 ) and cloned into pLV-EF1α-IRES-Puro vector (Addgene #85132).

Techniques: Virus, Binding Assay, Incubation, Quantitative RT-PCR, Control, Infection, Flow Cytometry, Staining, Transfection, Expressing, Activity Assay, Over Expression, Mutagenesis, Fluorescence, Microscopy

Journal: mBio

Article Title: Soluble MFGE8 mediates cell entry of Crimean-Congo hemorrhagic fever virus

doi: 10.1128/mbio.01617-25

Figure Lengend Snippet: MFGE8 directly binds to the Gc domain of CCHFV. ( A ) The structure of MFGE8 protein predicted by AlphaFold2. Three domains, EGF, C1, C2, are indicated. ( B ) Schematic of different truncations of MFGE8 protein. ( C ) Overexpression of different truncated forms of MFGE8 in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 20 h) and flow cytometry analysis. ( D ) Schematic of chimeric MFGE8 proteins. The full-length MFGE8 without signal peptide was fused with the type II transmembrane protein, TMPRSS2. The C1 and C2 domains of MFGE8 were fused with type I transmembrane protein, MXRA8. The extracellular region of TMPRSS13 or MXRA8 was replaced. ( E ). Overexpression of chimeric MFGE8 proteins in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 20 h) and flow cytometry analysis. ( F ) Overexpression of different mutants of MFGE8 in A549-BAT cells, followed by infection with rVSV-CCHFV (MOI 3, 20 h) and flow cytometry analysis. One-way ANOVA with Dunnett’s multiple comparisons test (C, E, and F). ns, not significant; * P < 0.1; *** P < 0.001; **** P < 0.0001. ( G ) Binding of MFGE8 protein to CCHFV Gc monomer, Gn, and Mpox virus B6 control protein. These proteins were pre-coated onto ELISA plates at a concentration of 10 µg/mL, followed by incubation of different concentrations of MFGE8 protein. The binding was evaluated using anti-MFGE8 and HRP-conjugated secondary antibodies. ( H–K ) MFGE8 and B6 control proteins were coated onto ELISA plates at a concentration of 10 µg/mL. Then, different concentrations of CCHFV Gc monomer ( H ), Gc trimer ( I ), or rVSV-CCHFV particles ( J ) were incubated and detected using anti-CCHFV-Gc ADI-36121 antibody and HRP-conjugated secondary antibody. ( K ) The binding affinity of MFGE8 to CCHFV Gc domain. His-tagged MFGE8 protein was immobilized onto the Ni-NTA biosensors. Binding parameters of recombinant Gc monomer to MFGE8 protein were measured by biolayer interferometry (BLI). Using a 1/1 binding model to derive equilibrium dissociation constant (Kd) values. ( L ) The model of soluble MFGE8-mediated cell entry of CCHFV. The soluble MFGE8 protein binds directly to both Gc protein and phosphatidylserine (PtdSer) on the surface of CCHFV virions and acts as a bridge by binding to the integrins on the cell membrane to promote virus infection.

Article Snippet: To construct the overexpressing cells, human MFGE8 cDNA was purchased from SinoBiological ( NM_005928.1 ) and cloned into pLV-EF1α-IRES-Puro vector (Addgene #85132).

Techniques: Over Expression, Infection, Flow Cytometry, Binding Assay, Virus, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Recombinant, Membrane