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Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry.

Lin LT, Chen TY, Lin SC, Chung CY, Lin TC, Wang GH, Anderson R, Lin CC, Richardson CD - BMC Microbiol. (2013)

Bottom Line: These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs).CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity.Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

ABSTRACT

Background: We previously identified two hydrolyzable tannins, chebulagic acid (CHLA) and punicalagin (PUG) that blocked herpes simplex virus type 1 (HSV-1) entry and spread. These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs). Based on this property, we evaluated their antiviral efficacy against several different viruses known to employ GAGs for host cell entry.

Results: Extensive analysis of the tannins' mechanism of action was performed on a panel of viruses during the attachment and entry steps of infection. Virus-specific binding assays and the analysis of viral spread during treatment with these compounds were also conducted. CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity. Moreover, the natural compounds inhibited viral attachment, penetration, and spread, to different degrees for each virus. Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses.

Conclusions: CHLA and PUG may be of value as broad-spectrum antivirals for limiting emerging/recurring viruses known to engage host cell GAGs for entry. Further studies testing the efficacy of these tannins in vivo against certain viruses are justified.

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Related in: MedlinePlus

Effects of CHLA and PUG against virus binding analyzed by ELISA. Different cell monolayers were pre-chilled at 4°C for 1 h and then inoculated with the respective viruses in the presence or absence of various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the cell monolayers were washed to remove unadsorbed virus, subsequently fixed with 4% PFA, and then blocked with 5% BSA. ELISA was performed with virus-specific antibodies and HRP-conjugated IgG, followed by development with a TMB substrate kit. The absorbance was immediately determined at 450 nm and values are expressed as the fold change of absorbance relative to the mock infection control (cells + DMSO), which is indicated by the dashed line. (A) Schematic of the experiment with the virus concentration (MOI) and test compound treatment time (i) indicated for each virus in the associated table. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results shown are means ± SEM from three independent experiments. See text for details.
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Figure 5: Effects of CHLA and PUG against virus binding analyzed by ELISA. Different cell monolayers were pre-chilled at 4°C for 1 h and then inoculated with the respective viruses in the presence or absence of various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the cell monolayers were washed to remove unadsorbed virus, subsequently fixed with 4% PFA, and then blocked with 5% BSA. ELISA was performed with virus-specific antibodies and HRP-conjugated IgG, followed by development with a TMB substrate kit. The absorbance was immediately determined at 450 nm and values are expressed as the fold change of absorbance relative to the mock infection control (cells + DMSO), which is indicated by the dashed line. (A) Schematic of the experiment with the virus concentration (MOI) and test compound treatment time (i) indicated for each virus in the associated table. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results shown are means ± SEM from three independent experiments. See text for details.

Mentions: In method 2 (Figure 5A), different cell types (2 × 104 cells/well) were seeded in 96-well plates and grown overnight. The cell monolayers were pre-chilled at 4°C for 1 h and then co-treated with the respective viruses (HCMV, MOI = 5; HCV, MOI = 0.1; DENV-2, MOI = 5; MV, MOI = 1; RSV, MOI = 5) and various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the inocula and drugs were removed and the cell monolayers were washed with ice-cold PBS before fixation with pre-chilled 4% paraformaldehyde (PFA) in PBS for 1 h on ice. At that point, the wells were blocked with 5% bovine serum albumin (BSA) at 4°C overnight to prevent any non-specific binding. Bound viruses on the cellular surfaces were then detected by ELISA assay whereby wells were incubated with the following respective mouse monoclonal primary antibodies (diluted in PBS containing 5% BSA) at 37°C for 1 h before washing with PBST (0.1% Tween 20 in PBS) three times: anti-HCMV gB antibody (1:10,000; Thermo Pierce, Rockford, IL, USA), anti-HCV E2 antibody (1:20,000; AUSTRAL Biologicals, San Ramon, CA, USA), anti-flavivirus group antibody (1:5,000) for DENV-2, anti-measles hemagglutinin antibody (1:5,000; Millipore), and anti-RSV fusion protein antibody (1:15,000). Samples were then subjected to incubation at 37°C for 1 h with goat anti-mouse IgG conjugated with horseradish peroxidase (HRP; Invitrogen), diluted at 1:20,000 (HCMV, DENV-2, MV-EGFP), 1:36,000 (HCV), or 1:30,000 (RSV) in PBS containing 5% BSA. The wells were afterwards washed with PBST three times and developed with a TMB (3,3′,5,5′-tetramethylbenzidine) Two-component Microwell Peroxidase Substrate Kit (KPL, Gaithersburg, MD) at room temperature for 20 min before stopping the reaction with 1 M phosphoric acid (H3PO4). The plates were measured with an ELx800 Microplate reader (Instrument, Inc.; Winooski, VT, USA) at 450 nm.


Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry.

Lin LT, Chen TY, Lin SC, Chung CY, Lin TC, Wang GH, Anderson R, Lin CC, Richardson CD - BMC Microbiol. (2013)

Effects of CHLA and PUG against virus binding analyzed by ELISA. Different cell monolayers were pre-chilled at 4°C for 1 h and then inoculated with the respective viruses in the presence or absence of various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the cell monolayers were washed to remove unadsorbed virus, subsequently fixed with 4% PFA, and then blocked with 5% BSA. ELISA was performed with virus-specific antibodies and HRP-conjugated IgG, followed by development with a TMB substrate kit. The absorbance was immediately determined at 450 nm and values are expressed as the fold change of absorbance relative to the mock infection control (cells + DMSO), which is indicated by the dashed line. (A) Schematic of the experiment with the virus concentration (MOI) and test compound treatment time (i) indicated for each virus in the associated table. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results shown are means ± SEM from three independent experiments. See text for details.
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Related In: Results  -  Collection

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Figure 5: Effects of CHLA and PUG against virus binding analyzed by ELISA. Different cell monolayers were pre-chilled at 4°C for 1 h and then inoculated with the respective viruses in the presence or absence of various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the cell monolayers were washed to remove unadsorbed virus, subsequently fixed with 4% PFA, and then blocked with 5% BSA. ELISA was performed with virus-specific antibodies and HRP-conjugated IgG, followed by development with a TMB substrate kit. The absorbance was immediately determined at 450 nm and values are expressed as the fold change of absorbance relative to the mock infection control (cells + DMSO), which is indicated by the dashed line. (A) Schematic of the experiment with the virus concentration (MOI) and test compound treatment time (i) indicated for each virus in the associated table. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results shown are means ± SEM from three independent experiments. See text for details.
Mentions: In method 2 (Figure 5A), different cell types (2 × 104 cells/well) were seeded in 96-well plates and grown overnight. The cell monolayers were pre-chilled at 4°C for 1 h and then co-treated with the respective viruses (HCMV, MOI = 5; HCV, MOI = 0.1; DENV-2, MOI = 5; MV, MOI = 1; RSV, MOI = 5) and various concentrations of test compounds at 4°C for an additional 2 h. Following the virus binding period, the inocula and drugs were removed and the cell monolayers were washed with ice-cold PBS before fixation with pre-chilled 4% paraformaldehyde (PFA) in PBS for 1 h on ice. At that point, the wells were blocked with 5% bovine serum albumin (BSA) at 4°C overnight to prevent any non-specific binding. Bound viruses on the cellular surfaces were then detected by ELISA assay whereby wells were incubated with the following respective mouse monoclonal primary antibodies (diluted in PBS containing 5% BSA) at 37°C for 1 h before washing with PBST (0.1% Tween 20 in PBS) three times: anti-HCMV gB antibody (1:10,000; Thermo Pierce, Rockford, IL, USA), anti-HCV E2 antibody (1:20,000; AUSTRAL Biologicals, San Ramon, CA, USA), anti-flavivirus group antibody (1:5,000) for DENV-2, anti-measles hemagglutinin antibody (1:5,000; Millipore), and anti-RSV fusion protein antibody (1:15,000). Samples were then subjected to incubation at 37°C for 1 h with goat anti-mouse IgG conjugated with horseradish peroxidase (HRP; Invitrogen), diluted at 1:20,000 (HCMV, DENV-2, MV-EGFP), 1:36,000 (HCV), or 1:30,000 (RSV) in PBS containing 5% BSA. The wells were afterwards washed with PBST three times and developed with a TMB (3,3′,5,5′-tetramethylbenzidine) Two-component Microwell Peroxidase Substrate Kit (KPL, Gaithersburg, MD) at room temperature for 20 min before stopping the reaction with 1 M phosphoric acid (H3PO4). The plates were measured with an ELx800 Microplate reader (Instrument, Inc.; Winooski, VT, USA) at 450 nm.

Bottom Line: These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs).CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity.Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.

ABSTRACT

Background: We previously identified two hydrolyzable tannins, chebulagic acid (CHLA) and punicalagin (PUG) that blocked herpes simplex virus type 1 (HSV-1) entry and spread. These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs). Based on this property, we evaluated their antiviral efficacy against several different viruses known to employ GAGs for host cell entry.

Results: Extensive analysis of the tannins' mechanism of action was performed on a panel of viruses during the attachment and entry steps of infection. Virus-specific binding assays and the analysis of viral spread during treatment with these compounds were also conducted. CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity. Moreover, the natural compounds inhibited viral attachment, penetration, and spread, to different degrees for each virus. Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses.

Conclusions: CHLA and PUG may be of value as broad-spectrum antivirals for limiting emerging/recurring viruses known to engage host cell GAGs for entry. Further studies testing the efficacy of these tannins in vivo against certain viruses are justified.

Show MeSH
Related in: MedlinePlus