Limits...
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.

Show MeSH

Related in: MedlinePlus

Gal-1 acts at early stages during DENV-1 infection.(A) Binding of DENV-1 to hrGal-1 in a dose-dependent manner. Serial two-fold dilutions of DENV-1 were applied to 96-well plates coated with 1 µg of hrGal-1 per well, and the bound virus particles were detected by ELISA with mouse anti-E protein antibody. BSA-coated wells served as the negative control. To assess the involvement of Gal-1 CRD, the assay was performed in presence of 40 mM lactose or sucrose. Each value represents the mean±the SD from 4 assays performed in duplicates. (B) Adsorption and internalization assays: for adsorption assay, ECV-304 cells were infected with DENV-1 at MOI of 10 in presence or absence of 10 µM hrGal-1 during 1 h at 4°C and then washed to remove viral inoculum. Cells were collected and the viral RNA was quantified by Real-Time PCR. Data was normalized by host β-actin expression. For internalization assay, ECV-304 cells were inoculated with DENV-1 (MOI of 10) at 4°C for 1 hour. Then, cells were washed and transferred to 37°C and hrGal-1 (10 µM) or only medium were added to culture. After 1 hour of incubation, non-internalized viruses were inactivated with citrate buffer and viral loads were quantified by Real-Time PCR. Data is presented as Viral RNA amount equivalents to PFU/mL±SD from 3 experiments assessed in triplicates. (C) For virucidal assay, DENV-1 was incubated with hrGal-1, in the presence or absence of RNAse. After 1 h incubation at 37°C, RNA was isolated and subjected to RT-Real-Time PCR. Purified viral RNA incubated or not with RNAse was used as control (N = 3). (D) ECV-304 cells were infected with DENV-1 at a MOI of 0.5 (DENV-1). For the treatments, hrGal-1 was incubated with ECV-304 and DENV-1 simultaneously (ECV+Gal-1+DENV), or hrGal-1 (10 µM) was pre-incubated with either ECV-304 cells or with DENV-1 (MOI 0.5) for 60 minutes before the inoculation (ECV+hrGal-1)+DENV versus (DENV+hrGal-1)+ECV, respectively. At 72 hours postinfection, supernatants were collected and the viral loads were quantified by Real-Time PCR (N = 3). **p<0.001; ***p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4231055&req=5

pone-0112474-g004: Gal-1 acts at early stages during DENV-1 infection.(A) Binding of DENV-1 to hrGal-1 in a dose-dependent manner. Serial two-fold dilutions of DENV-1 were applied to 96-well plates coated with 1 µg of hrGal-1 per well, and the bound virus particles were detected by ELISA with mouse anti-E protein antibody. BSA-coated wells served as the negative control. To assess the involvement of Gal-1 CRD, the assay was performed in presence of 40 mM lactose or sucrose. Each value represents the mean±the SD from 4 assays performed in duplicates. (B) Adsorption and internalization assays: for adsorption assay, ECV-304 cells were infected with DENV-1 at MOI of 10 in presence or absence of 10 µM hrGal-1 during 1 h at 4°C and then washed to remove viral inoculum. Cells were collected and the viral RNA was quantified by Real-Time PCR. Data was normalized by host β-actin expression. For internalization assay, ECV-304 cells were inoculated with DENV-1 (MOI of 10) at 4°C for 1 hour. Then, cells were washed and transferred to 37°C and hrGal-1 (10 µM) or only medium were added to culture. After 1 hour of incubation, non-internalized viruses were inactivated with citrate buffer and viral loads were quantified by Real-Time PCR. Data is presented as Viral RNA amount equivalents to PFU/mL±SD from 3 experiments assessed in triplicates. (C) For virucidal assay, DENV-1 was incubated with hrGal-1, in the presence or absence of RNAse. After 1 h incubation at 37°C, RNA was isolated and subjected to RT-Real-Time PCR. Purified viral RNA incubated or not with RNAse was used as control (N = 3). (D) ECV-304 cells were infected with DENV-1 at a MOI of 0.5 (DENV-1). For the treatments, hrGal-1 was incubated with ECV-304 and DENV-1 simultaneously (ECV+Gal-1+DENV), or hrGal-1 (10 µM) was pre-incubated with either ECV-304 cells or with DENV-1 (MOI 0.5) for 60 minutes before the inoculation (ECV+hrGal-1)+DENV versus (DENV+hrGal-1)+ECV, respectively. At 72 hours postinfection, supernatants were collected and the viral loads were quantified by Real-Time PCR (N = 3). **p<0.001; ***p<0.0001.

Mentions: To further investigate the inhibitory effect of exogenous Gal-1 on DENV-1 infection, we first checked whether Gal-1 was able to bind directly to the virus and exert any direct virucidal activity against DENV-1. As shown in Figure 4A, DENV-1 bound to immobilized hrGal-1 in a dose-dependent manner, whereas the virus failed to bind to BSA-coated wells. Unexpectedly, the binding of DENV-1 to hrGal-1 was not abolished by the addition of lactose, an inhibitor of carbohydrate binding (Figure 4A). This may mean that the affinity of binding between hrGal-1 and DENV-1 is extremely high toward immobilized hrGal-1, and thus not readily reversible by lactose, or binding occurs through a non-carbohydrate interaction to hrGal-1. We next evaluated whether hrGal-1 could affect virus adsorption and/or virus internalization. As shown in Figure 4B, hrGal-1 significantly inhibited virus adsorption at 4°C for 1 hour (Figure 4B) when hrGal-1 was present during the incubation. If adsorption took place in a hrGal-1-free medium, and the lectin was added at culture supernatants when the temperature was raised at 37°C and maintained only additional 1 h of incubation, the virus yield also significantly decreased in comparison with untreated cultures (Figure 4B). A possible virucidal effect of hrGal-1 on DENV-1 was discarded by a virucidal assay (Figure 4C). Altogether, these results suggest that hrGal-1 may influence the early steps of DENV-1 infection. Finally, we investigated whether the inhibitory effects exerted by Gal-1 also depended on its interaction with the target cells. The pretreatment of DENV-1 with hrGal-1 [(DENV1+rhGal-1)+ECV] led to a significant decrease in viral load in the culture supernatants (Figure 4D). However, when ECV-304 cells were treated with hrGal-1 before DENV infection [(ECV+rhGal-1)+DENV1] or ECV-304 cells were treated concomitantly with hrGal-1 and DENV-1 (ECV+rhGal-1+DENV1), we observed an even greater reduction in the viral load in the culture supernatants (Figure 4D).


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)

Gal-1 acts at early stages during DENV-1 infection.(A) Binding of DENV-1 to hrGal-1 in a dose-dependent manner. Serial two-fold dilutions of DENV-1 were applied to 96-well plates coated with 1 µg of hrGal-1 per well, and the bound virus particles were detected by ELISA with mouse anti-E protein antibody. BSA-coated wells served as the negative control. To assess the involvement of Gal-1 CRD, the assay was performed in presence of 40 mM lactose or sucrose. Each value represents the mean±the SD from 4 assays performed in duplicates. (B) Adsorption and internalization assays: for adsorption assay, ECV-304 cells were infected with DENV-1 at MOI of 10 in presence or absence of 10 µM hrGal-1 during 1 h at 4°C and then washed to remove viral inoculum. Cells were collected and the viral RNA was quantified by Real-Time PCR. Data was normalized by host β-actin expression. For internalization assay, ECV-304 cells were inoculated with DENV-1 (MOI of 10) at 4°C for 1 hour. Then, cells were washed and transferred to 37°C and hrGal-1 (10 µM) or only medium were added to culture. After 1 hour of incubation, non-internalized viruses were inactivated with citrate buffer and viral loads were quantified by Real-Time PCR. Data is presented as Viral RNA amount equivalents to PFU/mL±SD from 3 experiments assessed in triplicates. (C) For virucidal assay, DENV-1 was incubated with hrGal-1, in the presence or absence of RNAse. After 1 h incubation at 37°C, RNA was isolated and subjected to RT-Real-Time PCR. Purified viral RNA incubated or not with RNAse was used as control (N = 3). (D) ECV-304 cells were infected with DENV-1 at a MOI of 0.5 (DENV-1). For the treatments, hrGal-1 was incubated with ECV-304 and DENV-1 simultaneously (ECV+Gal-1+DENV), or hrGal-1 (10 µM) was pre-incubated with either ECV-304 cells or with DENV-1 (MOI 0.5) for 60 minutes before the inoculation (ECV+hrGal-1)+DENV versus (DENV+hrGal-1)+ECV, respectively. At 72 hours postinfection, supernatants were collected and the viral loads were quantified by Real-Time PCR (N = 3). **p<0.001; ***p<0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231055&req=5

pone-0112474-g004: Gal-1 acts at early stages during DENV-1 infection.(A) Binding of DENV-1 to hrGal-1 in a dose-dependent manner. Serial two-fold dilutions of DENV-1 were applied to 96-well plates coated with 1 µg of hrGal-1 per well, and the bound virus particles were detected by ELISA with mouse anti-E protein antibody. BSA-coated wells served as the negative control. To assess the involvement of Gal-1 CRD, the assay was performed in presence of 40 mM lactose or sucrose. Each value represents the mean±the SD from 4 assays performed in duplicates. (B) Adsorption and internalization assays: for adsorption assay, ECV-304 cells were infected with DENV-1 at MOI of 10 in presence or absence of 10 µM hrGal-1 during 1 h at 4°C and then washed to remove viral inoculum. Cells were collected and the viral RNA was quantified by Real-Time PCR. Data was normalized by host β-actin expression. For internalization assay, ECV-304 cells were inoculated with DENV-1 (MOI of 10) at 4°C for 1 hour. Then, cells were washed and transferred to 37°C and hrGal-1 (10 µM) or only medium were added to culture. After 1 hour of incubation, non-internalized viruses were inactivated with citrate buffer and viral loads were quantified by Real-Time PCR. Data is presented as Viral RNA amount equivalents to PFU/mL±SD from 3 experiments assessed in triplicates. (C) For virucidal assay, DENV-1 was incubated with hrGal-1, in the presence or absence of RNAse. After 1 h incubation at 37°C, RNA was isolated and subjected to RT-Real-Time PCR. Purified viral RNA incubated or not with RNAse was used as control (N = 3). (D) ECV-304 cells were infected with DENV-1 at a MOI of 0.5 (DENV-1). For the treatments, hrGal-1 was incubated with ECV-304 and DENV-1 simultaneously (ECV+Gal-1+DENV), or hrGal-1 (10 µM) was pre-incubated with either ECV-304 cells or with DENV-1 (MOI 0.5) for 60 minutes before the inoculation (ECV+hrGal-1)+DENV versus (DENV+hrGal-1)+ECV, respectively. At 72 hours postinfection, supernatants were collected and the viral loads were quantified by Real-Time PCR (N = 3). **p<0.001; ***p<0.0001.
Mentions: To further investigate the inhibitory effect of exogenous Gal-1 on DENV-1 infection, we first checked whether Gal-1 was able to bind directly to the virus and exert any direct virucidal activity against DENV-1. As shown in Figure 4A, DENV-1 bound to immobilized hrGal-1 in a dose-dependent manner, whereas the virus failed to bind to BSA-coated wells. Unexpectedly, the binding of DENV-1 to hrGal-1 was not abolished by the addition of lactose, an inhibitor of carbohydrate binding (Figure 4A). This may mean that the affinity of binding between hrGal-1 and DENV-1 is extremely high toward immobilized hrGal-1, and thus not readily reversible by lactose, or binding occurs through a non-carbohydrate interaction to hrGal-1. We next evaluated whether hrGal-1 could affect virus adsorption and/or virus internalization. As shown in Figure 4B, hrGal-1 significantly inhibited virus adsorption at 4°C for 1 hour (Figure 4B) when hrGal-1 was present during the incubation. If adsorption took place in a hrGal-1-free medium, and the lectin was added at culture supernatants when the temperature was raised at 37°C and maintained only additional 1 h of incubation, the virus yield also significantly decreased in comparison with untreated cultures (Figure 4B). A possible virucidal effect of hrGal-1 on DENV-1 was discarded by a virucidal assay (Figure 4C). Altogether, these results suggest that hrGal-1 may influence the early steps of DENV-1 infection. Finally, we investigated whether the inhibitory effects exerted by Gal-1 also depended on its interaction with the target cells. The pretreatment of DENV-1 with hrGal-1 [(DENV1+rhGal-1)+ECV] led to a significant decrease in viral load in the culture supernatants (Figure 4D). However, when ECV-304 cells were treated with hrGal-1 before DENV infection [(ECV+rhGal-1)+DENV1] or ECV-304 cells were treated concomitantly with hrGal-1 and DENV-1 (ECV+rhGal-1+DENV1), we observed an even greater reduction in the viral load in the culture supernatants (Figure 4D).

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