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Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model.

Aerts L, Hamelin MÈ, Rhéaume C, Lavigne S, Couture C, Kim W, Susan-Resiga D, Prat A, Seidah NG, Vergnolle N, Riteau B, Boivin G - PLoS ONE (2013)

Bottom Line: Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs.Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time.Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Infectiologie du Centre Hospitalier Universitaire de Québec and Université Laval, Quebec, Canada.

ABSTRACT
Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.

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Effect of PAR1 agonist or antagonist treatment of hMPV-infected mice on furin expression.A) Groups of 6 mice were infected intranasally with hMPV (7x105 TCID50) or mock infected and simultaneously treated for 5 days with a single daily dose of 500 µM of PAR1 agonist (TFLLR-NH2) or PAR1 antagonist (SCH79797). Mice were sacrificed on day 5 pi, lungs were removed and snap frozen. RNA was extracted and furin transcript levels were determined using RT-PCR. Significant differences in furin transcript levels were observed between treated and untreated mice based on a Student T-test (* p=0.05, ** p<0.01) B) COS-1 and HEK293 cells were co-transfected with a plasmid encoding the hMPV F protein containing a V5-tag and a plasmid encoding one of the proprotein convertases. Cell lysates were analyzed by western blot using a V5 mAb. Furin is the only convertase capable of cleaving the full length precursor protein (F0) into its activated form, resulting in the shorter C-terminal subunit (F1). C) Recombinant human PAR1 and furin are co-tranfected with the hMPV F protein containing a V5-tag in HEK293 cells with/without hPAR1 agonist, (100 μM) or antagonist (0.1 μM). Western blot analysis of cell lysates using an anti-V5 mAb is shown.
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pone-0072529-g004: Effect of PAR1 agonist or antagonist treatment of hMPV-infected mice on furin expression.A) Groups of 6 mice were infected intranasally with hMPV (7x105 TCID50) or mock infected and simultaneously treated for 5 days with a single daily dose of 500 µM of PAR1 agonist (TFLLR-NH2) or PAR1 antagonist (SCH79797). Mice were sacrificed on day 5 pi, lungs were removed and snap frozen. RNA was extracted and furin transcript levels were determined using RT-PCR. Significant differences in furin transcript levels were observed between treated and untreated mice based on a Student T-test (* p=0.05, ** p<0.01) B) COS-1 and HEK293 cells were co-transfected with a plasmid encoding the hMPV F protein containing a V5-tag and a plasmid encoding one of the proprotein convertases. Cell lysates were analyzed by western blot using a V5 mAb. Furin is the only convertase capable of cleaving the full length precursor protein (F0) into its activated form, resulting in the shorter C-terminal subunit (F1). C) Recombinant human PAR1 and furin are co-tranfected with the hMPV F protein containing a V5-tag in HEK293 cells with/without hPAR1 agonist, (100 μM) or antagonist (0.1 μM). Western blot analysis of cell lysates using an anti-V5 mAb is shown.

Mentions: The proprotein convertases, especially furin, have been shown to process a number of cell surface glycoproteins of infectious viruses both at single and paired basic residues. The minimal cleavage site is RXXR↓, exhibiting a P1 and P4 Arg residues [46]. In order to tentatively investigate the possible mechanism by which PAR1 compounds can influence viral replication, we analyzed furin transcripts in the lungs of hMPV-infected animals by RT-PCR. PAR1 compounds were administered for 5 days at a dose of 500 µM, starting at the time of infection, and lungs were harvested on day 5 pi. HMPV infection resulted in a significant increase in furin transcripts (by 38%) in mouse lungs compared to uninfected mice (Fig. 4A). Treatment of infected mice with the PAR1 agonist did not significantly alter furin expression. However, treatment of infected mice with the PAR1 antagonist significantly reduced furin transcript levels (by 18%) compared to hMPV-infected/untreated mice. Using a pIRES vector expressing each proprotein convertase, we previously showed a similar expression of each convertase and their ability to cleave selected substrates [47,48]. Thus, we used these same constructs to show that furin was the only tested proprotein convertase that was able to cleave the hMPV fusion precursor protein into its active form (Figure 4B) in two cell lines (COS-1 and HEK293). We then investigated the effect of PAR1 on the cleavage of the hMPV fusion precursor protein (Figure 4C) in this in vitro cleavage assay. Treatment of F protein/furin co-transfected HEK293 cells with either the PAR1 agonist or antagonist did not alter F cleavage (43.7 and 41.2% cleavage, respectively, compared to 36.8%). However, co-transfection of cells with cDNA encoding the F protein, furin as well as recombinant human PAR1 (rhPAR1) reduced F cleavage from 36.8% to 12.4%. When these rhPAR1 expressing co-transfected cells were treated with the PAR1 antagonist, F protein cleavage remained low (12.0%), whereas PAR1 agonist treatment restored F protein cleavage to the basal level observed in cells without rhPAR1 expression (38.2%), confirming a role for PAR1 in the cleavage of the hMPV fusion protein.


Modulation of protease activated receptor 1 influences human metapneumovirus disease severity in a mouse model.

Aerts L, Hamelin MÈ, Rhéaume C, Lavigne S, Couture C, Kim W, Susan-Resiga D, Prat A, Seidah NG, Vergnolle N, Riteau B, Boivin G - PLoS ONE (2013)

Effect of PAR1 agonist or antagonist treatment of hMPV-infected mice on furin expression.A) Groups of 6 mice were infected intranasally with hMPV (7x105 TCID50) or mock infected and simultaneously treated for 5 days with a single daily dose of 500 µM of PAR1 agonist (TFLLR-NH2) or PAR1 antagonist (SCH79797). Mice were sacrificed on day 5 pi, lungs were removed and snap frozen. RNA was extracted and furin transcript levels were determined using RT-PCR. Significant differences in furin transcript levels were observed between treated and untreated mice based on a Student T-test (* p=0.05, ** p<0.01) B) COS-1 and HEK293 cells were co-transfected with a plasmid encoding the hMPV F protein containing a V5-tag and a plasmid encoding one of the proprotein convertases. Cell lysates were analyzed by western blot using a V5 mAb. Furin is the only convertase capable of cleaving the full length precursor protein (F0) into its activated form, resulting in the shorter C-terminal subunit (F1). C) Recombinant human PAR1 and furin are co-tranfected with the hMPV F protein containing a V5-tag in HEK293 cells with/without hPAR1 agonist, (100 μM) or antagonist (0.1 μM). Western blot analysis of cell lysates using an anti-V5 mAb is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3755973&req=5

pone-0072529-g004: Effect of PAR1 agonist or antagonist treatment of hMPV-infected mice on furin expression.A) Groups of 6 mice were infected intranasally with hMPV (7x105 TCID50) or mock infected and simultaneously treated for 5 days with a single daily dose of 500 µM of PAR1 agonist (TFLLR-NH2) or PAR1 antagonist (SCH79797). Mice were sacrificed on day 5 pi, lungs were removed and snap frozen. RNA was extracted and furin transcript levels were determined using RT-PCR. Significant differences in furin transcript levels were observed between treated and untreated mice based on a Student T-test (* p=0.05, ** p<0.01) B) COS-1 and HEK293 cells were co-transfected with a plasmid encoding the hMPV F protein containing a V5-tag and a plasmid encoding one of the proprotein convertases. Cell lysates were analyzed by western blot using a V5 mAb. Furin is the only convertase capable of cleaving the full length precursor protein (F0) into its activated form, resulting in the shorter C-terminal subunit (F1). C) Recombinant human PAR1 and furin are co-tranfected with the hMPV F protein containing a V5-tag in HEK293 cells with/without hPAR1 agonist, (100 μM) or antagonist (0.1 μM). Western blot analysis of cell lysates using an anti-V5 mAb is shown.
Mentions: The proprotein convertases, especially furin, have been shown to process a number of cell surface glycoproteins of infectious viruses both at single and paired basic residues. The minimal cleavage site is RXXR↓, exhibiting a P1 and P4 Arg residues [46]. In order to tentatively investigate the possible mechanism by which PAR1 compounds can influence viral replication, we analyzed furin transcripts in the lungs of hMPV-infected animals by RT-PCR. PAR1 compounds were administered for 5 days at a dose of 500 µM, starting at the time of infection, and lungs were harvested on day 5 pi. HMPV infection resulted in a significant increase in furin transcripts (by 38%) in mouse lungs compared to uninfected mice (Fig. 4A). Treatment of infected mice with the PAR1 agonist did not significantly alter furin expression. However, treatment of infected mice with the PAR1 antagonist significantly reduced furin transcript levels (by 18%) compared to hMPV-infected/untreated mice. Using a pIRES vector expressing each proprotein convertase, we previously showed a similar expression of each convertase and their ability to cleave selected substrates [47,48]. Thus, we used these same constructs to show that furin was the only tested proprotein convertase that was able to cleave the hMPV fusion precursor protein into its active form (Figure 4B) in two cell lines (COS-1 and HEK293). We then investigated the effect of PAR1 on the cleavage of the hMPV fusion precursor protein (Figure 4C) in this in vitro cleavage assay. Treatment of F protein/furin co-transfected HEK293 cells with either the PAR1 agonist or antagonist did not alter F cleavage (43.7 and 41.2% cleavage, respectively, compared to 36.8%). However, co-transfection of cells with cDNA encoding the F protein, furin as well as recombinant human PAR1 (rhPAR1) reduced F cleavage from 36.8% to 12.4%. When these rhPAR1 expressing co-transfected cells were treated with the PAR1 antagonist, F protein cleavage remained low (12.0%), whereas PAR1 agonist treatment restored F protein cleavage to the basal level observed in cells without rhPAR1 expression (38.2%), confirming a role for PAR1 in the cleavage of the hMPV fusion protein.

Bottom Line: Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs.Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time.Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties.

View Article: PubMed Central - PubMed

Affiliation: Centre de Recherche en Infectiologie du Centre Hospitalier Universitaire de Québec and Université Laval, Quebec, Canada.

ABSTRACT
Human metapneumovirus (hMPV) infection causes acute respiratory tract infections (RTI) which can result in hospitalization of both children and adults. To date, no antiviral or vaccine is available for this common viral infection. Immunomodulators could represent an interesting strategy for the treatment of severe viral infection. Recently, the role of protease-activated receptors (PAR) in inflammation, coagulation and infection processes has been of growing interest. Herein, the effects of a PAR1 agonist and a PAR1 antagonist on hMPV infection were investigated in BALB/c mice. Intranasal administration of the PAR1 agonist resulted in increased weight loss and mortality of infected mice. Conversely, the PAR1 antagonist was beneficial to hMPV infection by decreasing weight loss and clinical signs and by significantly reducing pulmonary inflammation, pro-inflammatory cytokine levels (including IL-6, KC and MCP-1) and recruitment of immune cells to the lungs. In addition, a significant reduction in pulmonary viral titers was also observed in the lungs of PAR1 antagonist-treated mice. Despite no apparent direct effect on virus replication during in vitro experiments, an important role for PAR1 in the regulation of furin expression in the lungs was shown for the first time. Further experiments indicated that the hMPV fusion protein can be cleaved by furin thus suggesting that PAR1 could have an effect on viral infectivity in addition to its immunomodulatory properties. Thus, inhibition of PAR1 by selected antagonists could represent an interesting strategy for decreasing the severity of paramyxovirus infections.

Show MeSH
Related in: MedlinePlus