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

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.

Figure 1

Structures of chebulagic acid (CHLA) and punicalagin (PUG). The chemical structures of the two hydrolyzable tannins under study, chebulagic acid (CHLA) and punicalagin (PUG), are presented.

Figure 2

Dose response of CHLA and PUG treatments against multiple viruses. Host cells for each virus (HEL for HCMV; Huh-7.5 for HCV; Vero for DENV-2, CHO-SLAM for MV; HEp-2 for RSV, and A549 for VSV and ADV-5) were co-treated with viral inoculum and increasing concentrations of test compounds for 1 – 3 h before being washed, incubated, and analyzed for virus infection by plaque assays, EGFP expression analysis, or luciferase assay as described in Methods. (A) Schematic of the experiment (shown on the left) with the virus concentration (PFU/well or MOI), co-treatment time (i), and the subsequent viral incubation period (ii) indicated for each virus in the table on the right. (B) Antiviral effect of CHLA against multiple viruses. (C) Antiviral effect of PUG against multiple viruses. Results are plotted against values for the DMSO control treatment of virus infections and the data shown are means ± the standard errors of the mean (SEM) from three independent experiments. See text for details.

Figure 3

Inactivation of viral infections by CHLA and PUG. Different viruses were treated with the test compounds for a long period (incubated for 1.5 – 3 h before titration; light gray bars) or short period (immediately diluted; dark gray bars) at 37°C before diluting it 50 – 100 fold to sub-therapeutic concentrations and subsequent analysis of infection on the respective host cells. (A) Schematics of the experiment (shown on the left) with the final virus concentration (PFU/well or MOI), long-term virus-drug incubation period (i), and the subsequent incubation time (ii) indicated for each virus in the table on the right. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Results are plotted against the DMSO negative control treatment for virus infection and the data shown are the means ± SEM from three independent experiments. See text for details.

Figure 4

Evaluation of antiviral activities of CHLA and PUG that affect virus attachment and penetration. (A) Schematics of the experiments with the virus concentration (PFU/well or MOI) and the time of addition and treatment with tannins (i, ii, iii) for each virus in the associated tables. In virus attachment analysis by Method 1 (light gray bars), monolayers of different cell types were pre-chilled at 4°C for 1 h, and then co-treated with the respective viruses and test compounds at 4°C (1.5 – 3 h; i) before washing off the inoculates and test compounds for subsequent incubation (37°C; ii) and examination of virus infection. In virus penetration analysis (dark gray bars), seeded cell monolayers were pre-chilled at 4°C for 1 h and then challenged with the respective viruses at 4°C for 1.5 – 3 h (i). Cells were then washed and treated with the test compounds for an additional incubation period (ii) during which the temperature was shifted to 37°C to facilitate viral penetration. At the end of the incubation, extracellular viruses were removed by either citrate buffer (pH 3.0) or PBS washes and the cells were further incubated (iii) for analysis of virus infection. Results for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. Data are plotted against the DMSO negative control treatment of virus infection and are presented as means ± SEM from three independent experiments. See text for details.

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.

Figure 6

Post-infection analysis of antiviral effects due to CHLA and PUG. Cell monolayers were inoculated with the respective viruses at 37°C to allow viral entry, then washed by citrate buffer or PBS to remove extracellular viruses, and subsequently incubated in the presence or absence of the test compounds for infection analysis. (A) Schematic of the experiment (left) with the virus concentration (PFU/well or MOI), virus infection time (i), and test compound treatment period post-infection (ii) indicated for each virus in the table shown on the right. Results for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. IFN-α treatment was included as control for HCV infection. Data shown are means ± SEM from three independent experiments. See text for details.

Figure 7

Examination of CHLA and PUG treatment on virus cell-to-cell spread. (A) Schematic of the experiment (left) with the virus concentration (PFU/well) and step-wise incubation periods (i, ii, iii) indicated for each virus in the table on the right. Virus infections were established (i) in the different cell types by direct inoculation (HCMV, DENV-2, MV, and RSV) or electroporation of viral RNA (HCV; *), and the cell monolayers were washed with citrate buffer or PBS before being covered with an overlay medium that prevents secondary infection. Initial virus plaques were allowed to form in the subsequent infections (ii), and then the test compounds were added to the overlay medium for an additional time of incubation (iii) before analysis of viral plaque size by immune fluorescence microscopy. Five random virus-positive plaques at the endpoint of the experiment were evaluated for each treatment group of viruses, and the data was plotted as “fold change of plaque area” against the size of the initial viral plaques formed prior to test compound treatment. Analyses for (B) HCMV, (C) HCV, (D) DENV-2, (E) MV, and (F) RSV are indicated in each additional panel. The S29 cell line and the FIP inhibitor were included as controls for HCV and MV, respectively. Results shown are means ± SEM from three independent experiments and representative micrographs of the evaluated plaques are provided in Additional file 1 Figure S1, Additional file 2 Figure S2, Additional file 3 Figure S3, Additional file 4 Figure S4 and Additional file 5 Figure S5. See text for details.