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Malaysia/B HA is proteolytically activated by murine membrane-anchored proteases TMPRSS4, TMPRSS13, hepsin, and prostasin, but not <t>matriptase.</t> A, examination of HA cleavage. HEK293 cells were co-transfected with plasmids encoding Malaysia/B HA and either TMPRSS2, TMPRSS4, TMPRSS13, prostasin, hepsin, or matriptase. Co-transfection of HA-expressing plasmid and empty vector (w/o) and transfection of empty vector only (ev) were used as control. Cell lysates were subjected to SDS-PAGE and Western blot analysis at 48 h post-transfection using HA-specific antibodies. β-Actin served as loading control. B, proteolytic activation and multicycle replication analysis. MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with Malaysia/B at a MOI 0.01. At 24 h p.i., cells were fixed and immunostained against the viral NP to examine virus spread. Transfection of cells with empty vector (ev) was used as negative control. Data are representatives of at least three independent experiments. C, cleavage of H9 by matriptase. HEK293 cells were co-transfected with plasmids encoding H9 of A/quail/Shantou/782/00 (H9N2) and either TMPRSS2 or matriptase. Co-transfection of H9-expressing plasmid and empty vector (w/o) or transfection of empty vector only (ev) were used as control. Cell lysates were analyzed by SDS-PAGE and Western blotting at 48 h post-transfection using H9-specific antibodies. β-Actin served as loading control. D, MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with H9N2 at an MOI of 0.03 for 24 h to allow multicycle replication. Cells were fixed and immunostained against NP. Transfection of cells with empty vector (ev) was used as negative control. Data are representative of three independent experiments.
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Malaysia/B HA is proteolytically activated by murine membrane-anchored proteases TMPRSS4, TMPRSS13, hepsin, and prostasin, but not matriptase. A, examination of HA cleavage. HEK293 cells were co-transfected with plasmids encoding Malaysia/B HA and either TMPRSS2, TMPRSS4, TMPRSS13, prostasin, hepsin, or matriptase. Co-transfection of HA-expressing plasmid and empty vector (w/o) and transfection of empty vector only (ev) were used as control. Cell lysates were subjected to SDS-PAGE and Western blot analysis at 48 h post-transfection using HA-specific antibodies. β-Actin served as loading control. B, proteolytic activation and multicycle replication analysis. MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with Malaysia/B at a MOI 0.01. At 24 h p.i., cells were fixed and immunostained against the viral NP to examine virus spread. Transfection of cells with empty vector (ev) was used as negative control. Data are representatives of at least three independent experiments. C, cleavage of H9 by matriptase. HEK293 cells were co-transfected with plasmids encoding H9 of A/quail/Shantou/782/00 (H9N2) and either TMPRSS2 or matriptase. Co-transfection of H9-expressing plasmid and empty vector (w/o) or transfection of empty vector only (ev) were used as control. Cell lysates were analyzed by SDS-PAGE and Western blotting at 48 h post-transfection using H9-specific antibodies. β-Actin served as loading control. D, MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with H9N2 at an MOI of 0.03 for 24 h to allow multicycle replication. Cells were fixed and immunostained against NP. Transfection of cells with empty vector (ev) was used as negative control. Data are representative of three independent experiments.

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Malaysia/B HA is proteolytically activated by murine membrane-anchored proteases TMPRSS4, TMPRSS13, hepsin, and prostasin, but not matriptase. A, examination of HA cleavage. HEK293 cells were co-transfected with plasmids encoding Malaysia/B HA and either TMPRSS2, TMPRSS4, TMPRSS13, prostasin, hepsin, or matriptase. Co-transfection of HA-expressing plasmid and empty vector (w/o) and transfection of empty vector only (ev) were used as control. Cell lysates were subjected to SDS-PAGE and Western blot analysis at 48 h post-transfection using HA-specific antibodies. β-Actin served as loading control. B, proteolytic activation and multicycle replication analysis. MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with Malaysia/B at a MOI 0.01. At 24 h p.i., cells were fixed and immunostained against the viral NP to examine virus spread. Transfection of cells with empty vector (ev) was used as negative control. Data are representatives of at least three independent experiments. C, cleavage of H9 by matriptase. HEK293 cells were co-transfected with plasmids encoding H9 of A/quail/Shantou/782/00 (H9N2) and either TMPRSS2 or matriptase. Co-transfection of H9-expressing plasmid and empty vector (w/o) or transfection of empty vector only (ev) were used as control. Cell lysates were analyzed by SDS-PAGE and Western blotting at 48 h post-transfection using H9-specific antibodies. β-Actin served as loading control. D, MDCK cells were transfected with protease-encoding plasmids for 24 h and then infected with H9N2 at an MOI of 0.03 for 24 h to allow multicycle replication. Cells were fixed and immunostained against NP. Transfection of cells with empty vector (ev) was used as negative control. Data are representative of three independent experiments.

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Transfection, Cotransfection, Expressing, Plasmid Preparation, SDS Page, Western Blot, Activation Assay, Infection, Negative Control

Malaysia/B HA is not activated by murine tPA, uPA, LTF, NSP4, CFB, tryptase ϵ, and HGFA. A, examination of HA cleavage by soluble proteases present in cell supernatants. HEK293 cells with transient expression of Malaysia/B HA were incubated with cleared protease containing HEK293 cell supernatants as described under “Experimental procedures” (left) or recombinant rKLK8 (right). Treatment of HA-expressing cells with buffer (w/o) or trypsin was used as control. Cell lysates were analyzed for HA cleavage by immunoblotting. β-Actin was used as loading control. B, MDCK cells with transient protease expression were infected with Malaysia/B at a low MOI of 0.01 and incubated for 24 h to allow multicycle viral replication. Cells transfected with empty vector (ev) or murine TMPRSS2-expressing plasmid were used as control. Virus spread was visualized by immunostaining of infected cells against NP. C, expression analysis of tryptase ϵ_DDDDK mutant in HEK293 cells with or without enterokinase treatment. Supernatants of cells transfected with empty vector (ev) or tryptase ϵ_DDDDK-encoding plasmid were concentrated (5×) at 48 h post-transfection and analyzed by SDS-PAGE and Western blotting using tryptase ϵ–specific antibodies. Zymogen and mature form are indicated by filled and open arrowheads, respectively. D, examination of HA cleavage by tryptase ϵ. HEK293 cells expressing Malaysia/B HA were incubated with tryptase ϵ_DDDDK mutant–containing cell supernatants treated with or without enterokinase (10 IU). Treatment of HA-expressing cells with trypsin was used as control. Cell lysates were analyzed for HA cleavage. E, expression analysis of HGFA in HEK293 supernatants with and without matriptase treatment. At 48 h post-transfection with empty vector (ev) or HGFA-encoding plasmid cell supernatants were concentrated (5×), treated with or without matriptase (5.0 μg/ml) for 1 h at 37 °C, and analyzed by immunoblotting using a FLAG-specific antibody. Zymogen and mature form are indicated by filled and open arrowheads, respectively. F, examination of HA cleavage by HGFA. HEK293 cells co-transfected with plasmids encoding Malaysia/B HA and either empty vector (w/o) or HGFA-encoding plasmid were incubated with exogenous matriptase or trypsin (0.5 μg/ml each) or remained untreated for 24 h. Cell lysates were analyzed for HA cleavage by Western blotting.

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Malaysia/B HA is not activated by murine tPA, uPA, LTF, NSP4, CFB, tryptase ϵ, and HGFA. A, examination of HA cleavage by soluble proteases present in cell supernatants. HEK293 cells with transient expression of Malaysia/B HA were incubated with cleared protease containing HEK293 cell supernatants as described under “Experimental procedures” (left) or recombinant rKLK8 (right). Treatment of HA-expressing cells with buffer (w/o) or trypsin was used as control. Cell lysates were analyzed for HA cleavage by immunoblotting. β-Actin was used as loading control. B, MDCK cells with transient protease expression were infected with Malaysia/B at a low MOI of 0.01 and incubated for 24 h to allow multicycle viral replication. Cells transfected with empty vector (ev) or murine TMPRSS2-expressing plasmid were used as control. Virus spread was visualized by immunostaining of infected cells against NP. C, expression analysis of tryptase ϵ_DDDDK mutant in HEK293 cells with or without enterokinase treatment. Supernatants of cells transfected with empty vector (ev) or tryptase ϵ_DDDDK-encoding plasmid were concentrated (5×) at 48 h post-transfection and analyzed by SDS-PAGE and Western blotting using tryptase ϵ–specific antibodies. Zymogen and mature form are indicated by filled and open arrowheads, respectively. D, examination of HA cleavage by tryptase ϵ. HEK293 cells expressing Malaysia/B HA were incubated with tryptase ϵ_DDDDK mutant–containing cell supernatants treated with or without enterokinase (10 IU). Treatment of HA-expressing cells with trypsin was used as control. Cell lysates were analyzed for HA cleavage. E, expression analysis of HGFA in HEK293 supernatants with and without matriptase treatment. At 48 h post-transfection with empty vector (ev) or HGFA-encoding plasmid cell supernatants were concentrated (5×), treated with or without matriptase (5.0 μg/ml) for 1 h at 37 °C, and analyzed by immunoblotting using a FLAG-specific antibody. Zymogen and mature form are indicated by filled and open arrowheads, respectively. F, examination of HA cleavage by HGFA. HEK293 cells co-transfected with plasmids encoding Malaysia/B HA and either empty vector (w/o) or HGFA-encoding plasmid were incubated with exogenous matriptase or trypsin (0.5 μg/ml each) or remained untreated for 24 h. Cell lysates were analyzed for HA cleavage by Western blotting.

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Expressing, Incubation, Recombinant, Western Blot, Infection, Transfection, Plasmid Preparation, Immunostaining, Mutagenesis, SDS Page

Cleavage of IBV HA and H3 by protease candidates and confirmed protease activity

Journal: The Journal of Biological Chemistry

Article Title: Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways

doi: 10.1074/jbc.RA120.012635

Figure Lengend Snippet: Cleavage of IBV HA and H3 by protease candidates and confirmed protease activity

Article Snippet: The cDNA of the HA gene of B/Malaysia/2506/2004 was cloned from viral RNA by RT-PCR using HA-specific primers and subsequently subcloned into pCAGGS expression plasmid using EcoRI and NotI restriction sites. pCMV6-Entry expression plasmids encoding murine proteases with a C-terminal Myc-DDK tag were obtained from OriGene Technologies: CFB (MR210521), HGFA (MR216475), hepsin (MR219750), LTF (MR210170), tPA (MR208868), uPA (MR225747), tryptase ϵ (MR204321), NSP4 (MR217041), prostasin (MR223227), matriptase (MR222240), TMPRSS4 (MR206946), and TMPRSS13 (MR220731). pCAGGS plasmids encoding human or murine TMPRSS2 with C-terminal FLAG epitope have been described previously ( 6 , 15 ). pCAGGS encoding human prostasin was described previously ( 31 ). pcDNA6.2/C-emGFP-HPN encoding human hepsin (Human ORFeome Collaboration, Clone ID: 100004749) was generously provided by the Juha Klefström laboratory (University of Helsinki).

Techniques: Activity Assay