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Galectin-1 exerts inhibitory effects during DENV-1 infection.

Toledo KA, Fermino ML, Andrade Cdel C, Riul TB, Alves RT, Muller VD, Russo RR, Stowell SR, Cummings RD, Aquino VH, Dias-Baruffi M - PLoS ONE (2014)

Bottom Line: We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production.Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3.These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Universidade Estadual Paulista - UNESP (FCL-Assis), Assis, Brazil.

ABSTRACT
Dengue virus (DENV) is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1), we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1) inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.

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Treatment with human recombinant Gal-1 inhibits DENV-1 in vitro infection.(A) Biotinylated-hrGal-1 (20 µg/mL) was incubated with ECV-304 cells in presence or absence of 40 mM lactose or sucrose for 1 hour at 4°C. The binding of biotinylated-hrGal-1 to ECV-304 cells surfaces was detected by staining with streptavidin-FITC and measured by flow cytometry. The analysis was performed using a Diva software (Becton Dickson) and results are expressed as mean mean fluorescence intensity (MFI)±SD. Tests were performed in triplicates. (B) HMVEC-L, Vero-E6 and ECV-304 cells were incubated with 10 µM hrGal-1 or only medium for 1 hour at 37°C. Following, cells were inoculated with DENV-1 at a MOI of 0.5 and cultivated for 72 hours. At 72 hours postinfection the supernatants were collected and the viral RNA amounts were quantified by Real-Time PCR. Results are shown as Viral RNA amounts equivalent to PFU/ml±SD from 3 assays performed in triplicates. (C) ECV-304 cells were treated with hrGal-1 and infected with DENV-1 as described in (B) for 120 hours. The supernatants were collected at the indicated times postinfection and the viral loads were quantified by Real-Time PCR as described in (B). (N = 3) (D) ECV-304 cells were incubated with increased concentrations of monomeric-Gal-1 (hrGal-1 m), dimeric-Gal-1 (hrGal-1 d) or galectin-3 (hrGal-3) for 1 hour at 37°C before inoculation with DENV-1 (MOI 0.5). Cells were cultivated for 72 hours at 37°C and viral load was quantified as described in (A) (N = 3). (E) ECV-304 cells were treated with hrGal-1 (10 µM) in the presence of 40 mM lactose (LAC) or 40 mM sucrose (SUC) and then infected with DENV-1 as described in (B), for 72 hours. The viral load was quantified as described in (B). Data are representative from three independent experiments. *p<0.01; **p<0.001; ***p<0.0001.
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pone-0112474-g002: Treatment with human recombinant Gal-1 inhibits DENV-1 in vitro infection.(A) Biotinylated-hrGal-1 (20 µg/mL) was incubated with ECV-304 cells in presence or absence of 40 mM lactose or sucrose for 1 hour at 4°C. The binding of biotinylated-hrGal-1 to ECV-304 cells surfaces was detected by staining with streptavidin-FITC and measured by flow cytometry. The analysis was performed using a Diva software (Becton Dickson) and results are expressed as mean mean fluorescence intensity (MFI)±SD. Tests were performed in triplicates. (B) HMVEC-L, Vero-E6 and ECV-304 cells were incubated with 10 µM hrGal-1 or only medium for 1 hour at 37°C. Following, cells were inoculated with DENV-1 at a MOI of 0.5 and cultivated for 72 hours. At 72 hours postinfection the supernatants were collected and the viral RNA amounts were quantified by Real-Time PCR. Results are shown as Viral RNA amounts equivalent to PFU/ml±SD from 3 assays performed in triplicates. (C) ECV-304 cells were treated with hrGal-1 and infected with DENV-1 as described in (B) for 120 hours. The supernatants were collected at the indicated times postinfection and the viral loads were quantified by Real-Time PCR as described in (B). (N = 3) (D) ECV-304 cells were incubated with increased concentrations of monomeric-Gal-1 (hrGal-1 m), dimeric-Gal-1 (hrGal-1 d) or galectin-3 (hrGal-3) for 1 hour at 37°C before inoculation with DENV-1 (MOI 0.5). Cells were cultivated for 72 hours at 37°C and viral load was quantified as described in (A) (N = 3). (E) ECV-304 cells were treated with hrGal-1 (10 µM) in the presence of 40 mM lactose (LAC) or 40 mM sucrose (SUC) and then infected with DENV-1 as described in (B), for 72 hours. The viral load was quantified as described in (B). Data are representative from three independent experiments. *p<0.01; **p<0.001; ***p<0.0001.

Mentions: Since Gal-1 expression levels seemed to be inversely correlated to DENV permissiveness, we therefore evaluate whether the addition of exogenous Gal-1 could interfere with DENV infection in vitro. First, we demonstrated that ECV-304 cells display Gal-1-specific binding sites because human recombinant Gal-1 (hrGal-1) was able to bind ECV-304 cell surfaces, and this binding is abrogated in the presence of lactose, a weak but effective inhibitor of Gal-1, but not by sucrose, an isomeric sugar that does not bind galectins (Figure 2A). Similar results were obtained using HMVEC-L and Vero-E6 cells (data not shown). Monolayers of ECV-304, Vero-E6 and HMVEC-L cells were treated with 10 µM of hrGal-1 for 1 hour at 37°C, followed by addition DENV-1 virus (MOI 0.5) for 72 hours, and the viral load was quantified from the culture supernatants. As demonstrated before (Figure 1B), EVC-304 and Vero-E6 cells are much more permissive to DENV-1 infection than HMVEC-L cells; however, all the three cell lines had a significant reduction in viral load (35% in Vero-E6 cells, 60% in ECV-304 cells and 65% HMVEC-L cells) when pre-treated with hrGal-1 (Figure 2B). The kinetics of DENV-1 infection was also monitored in EVC-304 cells, pretreated or not with hrGal-1, during a period of 120 hours after infection. As shown in (Figure 2C), at 48, 72, 96 and 120 hours postinfection, the viral loads detected in the supernatant of DENV-1-infected ECV-304 cells pre-treated with hrGal-1 were significantly lower compared with viral load recovered from the supernatants of Gal-1-untreated cells. We also found a dose-dependent inhibition of DENV-1 infection when ECV-304 cells were pretreated with increasing hrGal-1 concentrations for 1 hour before the infection (Figure 2D).


Galectin-1 exerts inhibitory effects during DENV-1 infection.

Toledo KA, Fermino ML, Andrade Cdel C, Riul TB, Alves RT, Muller VD, Russo RR, Stowell SR, Cummings RD, Aquino VH, Dias-Baruffi M - PLoS ONE (2014)

Treatment with human recombinant Gal-1 inhibits DENV-1 in vitro infection.(A) Biotinylated-hrGal-1 (20 µg/mL) was incubated with ECV-304 cells in presence or absence of 40 mM lactose or sucrose for 1 hour at 4°C. The binding of biotinylated-hrGal-1 to ECV-304 cells surfaces was detected by staining with streptavidin-FITC and measured by flow cytometry. The analysis was performed using a Diva software (Becton Dickson) and results are expressed as mean mean fluorescence intensity (MFI)±SD. Tests were performed in triplicates. (B) HMVEC-L, Vero-E6 and ECV-304 cells were incubated with 10 µM hrGal-1 or only medium for 1 hour at 37°C. Following, cells were inoculated with DENV-1 at a MOI of 0.5 and cultivated for 72 hours. At 72 hours postinfection the supernatants were collected and the viral RNA amounts were quantified by Real-Time PCR. Results are shown as Viral RNA amounts equivalent to PFU/ml±SD from 3 assays performed in triplicates. (C) ECV-304 cells were treated with hrGal-1 and infected with DENV-1 as described in (B) for 120 hours. The supernatants were collected at the indicated times postinfection and the viral loads were quantified by Real-Time PCR as described in (B). (N = 3) (D) ECV-304 cells were incubated with increased concentrations of monomeric-Gal-1 (hrGal-1 m), dimeric-Gal-1 (hrGal-1 d) or galectin-3 (hrGal-3) for 1 hour at 37°C before inoculation with DENV-1 (MOI 0.5). Cells were cultivated for 72 hours at 37°C and viral load was quantified as described in (A) (N = 3). (E) ECV-304 cells were treated with hrGal-1 (10 µM) in the presence of 40 mM lactose (LAC) or 40 mM sucrose (SUC) and then infected with DENV-1 as described in (B), for 72 hours. The viral load was quantified as described in (B). Data are representative from three independent experiments. *p<0.01; **p<0.001; ***p<0.0001.
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pone-0112474-g002: Treatment with human recombinant Gal-1 inhibits DENV-1 in vitro infection.(A) Biotinylated-hrGal-1 (20 µg/mL) was incubated with ECV-304 cells in presence or absence of 40 mM lactose or sucrose for 1 hour at 4°C. The binding of biotinylated-hrGal-1 to ECV-304 cells surfaces was detected by staining with streptavidin-FITC and measured by flow cytometry. The analysis was performed using a Diva software (Becton Dickson) and results are expressed as mean mean fluorescence intensity (MFI)±SD. Tests were performed in triplicates. (B) HMVEC-L, Vero-E6 and ECV-304 cells were incubated with 10 µM hrGal-1 or only medium for 1 hour at 37°C. Following, cells were inoculated with DENV-1 at a MOI of 0.5 and cultivated for 72 hours. At 72 hours postinfection the supernatants were collected and the viral RNA amounts were quantified by Real-Time PCR. Results are shown as Viral RNA amounts equivalent to PFU/ml±SD from 3 assays performed in triplicates. (C) ECV-304 cells were treated with hrGal-1 and infected with DENV-1 as described in (B) for 120 hours. The supernatants were collected at the indicated times postinfection and the viral loads were quantified by Real-Time PCR as described in (B). (N = 3) (D) ECV-304 cells were incubated with increased concentrations of monomeric-Gal-1 (hrGal-1 m), dimeric-Gal-1 (hrGal-1 d) or galectin-3 (hrGal-3) for 1 hour at 37°C before inoculation with DENV-1 (MOI 0.5). Cells were cultivated for 72 hours at 37°C and viral load was quantified as described in (A) (N = 3). (E) ECV-304 cells were treated with hrGal-1 (10 µM) in the presence of 40 mM lactose (LAC) or 40 mM sucrose (SUC) and then infected with DENV-1 as described in (B), for 72 hours. The viral load was quantified as described in (B). Data are representative from three independent experiments. *p<0.01; **p<0.001; ***p<0.0001.
Mentions: Since Gal-1 expression levels seemed to be inversely correlated to DENV permissiveness, we therefore evaluate whether the addition of exogenous Gal-1 could interfere with DENV infection in vitro. First, we demonstrated that ECV-304 cells display Gal-1-specific binding sites because human recombinant Gal-1 (hrGal-1) was able to bind ECV-304 cell surfaces, and this binding is abrogated in the presence of lactose, a weak but effective inhibitor of Gal-1, but not by sucrose, an isomeric sugar that does not bind galectins (Figure 2A). Similar results were obtained using HMVEC-L and Vero-E6 cells (data not shown). Monolayers of ECV-304, Vero-E6 and HMVEC-L cells were treated with 10 µM of hrGal-1 for 1 hour at 37°C, followed by addition DENV-1 virus (MOI 0.5) for 72 hours, and the viral load was quantified from the culture supernatants. As demonstrated before (Figure 1B), EVC-304 and Vero-E6 cells are much more permissive to DENV-1 infection than HMVEC-L cells; however, all the three cell lines had a significant reduction in viral load (35% in Vero-E6 cells, 60% in ECV-304 cells and 65% HMVEC-L cells) when pre-treated with hrGal-1 (Figure 2B). The kinetics of DENV-1 infection was also monitored in EVC-304 cells, pretreated or not with hrGal-1, during a period of 120 hours after infection. As shown in (Figure 2C), at 48, 72, 96 and 120 hours postinfection, the viral loads detected in the supernatant of DENV-1-infected ECV-304 cells pre-treated with hrGal-1 were significantly lower compared with viral load recovered from the supernatants of Gal-1-untreated cells. We also found a dose-dependent inhibition of DENV-1 infection when ECV-304 cells were pretreated with increasing hrGal-1 concentrations for 1 hour before the infection (Figure 2D).

Bottom Line: We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production.Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3.These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Universidade Estadual Paulista - UNESP (FCL-Assis), Assis, Brazil.

ABSTRACT
Dengue virus (DENV) is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1), we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1) inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.

Show MeSH
Related in: MedlinePlus