Fumarprotocetraric acid and geraniin were identified as novel inhibitors of human respiratory syncytial virus infection in vitro

Abstract

Introduction: Respiratory syncytial virus (RSV) remains a major international public health concern. However, disease treatment is limited to preventive care with monoclonal antibodies and supportive care. In this study, natural products were screened to identify novel anti-RSV inhibitors.

Methods: The antiviral effect of 320 compounds on RSV in HEp-2 cells was tested using a Cytopathic effect (CPE) inhibition assay. The antiviral effect of fumarprotocetraric acid (FUM) and geraniin (GE) were confirmed by Real-time reverse transcription quantitative PCR (Real-time RT-PCR), plaque reduction test, immunofluorescence assay, and Western blot analysis. Real-time PCR was used to detect inflammatory factor expression. ATP assay and JC-1 stain were used to evaluate mitochondrial protection function. The experiment of administration time was used to determine the stages in the RSV life cycle inhibited by FUM and GE. Human metapneumovirus (HMPV) and human rhinovirus (HRV) were used to evaluate the antiviral activities of other respiratory viruses of FUM and GE. Finally, Air-liquid interface human airway epithelium (ALI-HAE) cells were used to evaluate the antiviral effect and mechanism of FUM and GE to RSV.

Results: The results showed that FUM and GE can inhibit the replication of RSV in multiple-cell models. Both compounds could dose-dependent inhibit the viral load, RSV nucleic acids level, and RSV-F protein level. Besides, FUM and GE showed good anti-inflammatory activity, mitochondrial protection, and antiviral activity to HMPV and HRV. Meanwhile, our result indicated that FUM and GE can inhibit RSV replication in ALI-HAE cells.

Conclusions: FUM and GE were identified as new inhibitors of RSV infection. At the same time, FUM and GE have anti-inflammatory activity, mitochondrial protection function, and broad-spectrum antiviral activity. These results provide evidence that FUM and GE are potential candidates for the development of novel anti-RSV drugs.

Keywords: antiviral; fumarprotocetraric acid; geraniin; natural products; respiratory syncytial virus.

Figure 1

Antiviral activity of fumarprotocetraric acid (FUM) and geraniin (GE) against RSV in cell-based assays. (A) Inhibit rate of 320 nature products. (B) Chemical structures of FUM and GE. (C) Concentration-inhibition response curve of FUM and GE against different RSV strains in HEp-2 cells. (D) The CPE images of FUM and GE against RSV Long strain in HEp-2 cells were obtained with the microscope (100×, scale bar = 100 μm).

Figure 2

Fumarprotocetraric acid (FUM) and geraniin (GE) inhibited virus replication in RSV-infected HEp-2 cells, A549 cells, and Vero cells. (A) RSV-M gene level detected by Real-time RT-PCR. (B) RSV viral titer was measured via plaque assay. Ribavirin group, 6.25μg/ml. (C) Representative image of plaque immunostaining results in wells at different dilutions. Virus plaque was labelled with anti-RSV-F antibody and HRP-conjugated secondary antibodies and then visualized with TrueBlue substrate (100×, scale bar = 100 μm). Data are described as the mean ± SEM (n = 3). *p < 0.05 and **p < 0.01, vs. the vehicle group.

Figure 3

Immunofluorescence detection of RSV-F protein expression inhibited by fumarprotocetraric acid (FUM) and geraniin (GE) in HEp-2 cells.

Figure 4

Fumarprotocetraric acid (FUM) and geraniin (GE) inhibited RSV-F protein expression assessed via Western blot in HEp-2 cells, A549 cells, and the Vero cells. Ribavirin group, 6.25μg/ml. Data are described as the mean ± SEM (n = 3). # p < 0.05 and ## p < 0.01, compared to the mock group. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the vehicle group.

Figure 5

Effect of fumarprotocetraric acid (FUM) and geraniin (GE) on cytokine production in RSV-infected cells. The transcription levels of mRNA of IL-1β, IL-4, IL-6, IL-8, TNF-α, and MCP-1 in HEp-2 cells were detected by Real-time PCR. Ribavirin group, 6.25μg/ml. Data are described as the mean ± SEM (n = 3). #### p < 0.0001, compared to the mock group. ***p < 0.001 and ****p < 0.0001 vs. the vehicle group.

Figure 6

The effects of fumarprotocetraric acid (FUM) and geraniin (GE) on mitochondrial function in RSV-infected cells. (A) The effects of FUM and GE on cell ATP level assessed by CellTiter-Glo Luminescent Cell Viability Assay in HEp-2 cells. (B, C) Mitochondrial Membrane Potential (MMP) Analysis of infected cells after FUM and GE treatment for 48h by flow cytometry analysis. Ribavirin: 6.25 μg/ml. Data are described as the mean ± SEM (n = 3). ## p< 0.01, and ### p< 0.001, compared to the mock group. *p < 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001 vs. the vehicle group.

Figure 7

Time of addition analysis of fumarprotocetraric acid (FUM) and geraniin (GE). (A) Schematic representation of the time-of-addition analysis. (B) The dose-effect curve under three administration times of FUM. (C) The dose-effect curve under three administration times of GE. Ribavirin: 6.25 μg/ml. Data are described as the mean ± SEM (n = 3).

Figure 8

Fumarprotocetraric acid (FUM) and geraniin (GE) inhibited respiratory virus in infected cell models. (A) The inhibiting effect of FUM and GE to HMPV-N gene nucleic acids level in LLC-MK2 detected by Real-time RT-PCR. (B) The inhibiting effect of FUM and GE to HRV-5’UTR nucleic acids level in H1-Hela detected by Real-time RT-PCR. (C) HRV CPE images were obtained with the microscope (100×, scale bar = 100 μm). Ribavirin group, 6.25μg/ml. Data are described as the mean ± SEM (n = 3). * p < 0.05 vs. the vehicle group.

Figure 9

Effect of fumarprotocetraric acid (FUM) and geraniin (GE) on virus replication in RSV-infected human airway epithelium (HAE) cells. (A) HAE cells were infected with RSV Long strain (0.01MOI). FUM and GE were added to the basolateral chamber of the Transwells (FUM and GE, 100μg/ml; Ribavirin, 6.25μg/ml). (B) Cytotoxic effects of FUM and GE on HAE cells detected by LDH. (C) Real-time RT-PCR detected collected virus RSV-M gene levels from the apical side. Data are described as the mean ± SEM (n = 3). *p < 0.05 and ****p < 0.0001 vs. the vehicle group.

Figure 10

Effects of fumarprotocetraric acid (FUM) and geraniin (GE) on differentially expressed genes (DEGs) in RSV-infected human airway epithelium (HAE). (A) The number of DEGs between each group. (B) Volcano map shows up-regulated and down-regulated DEGs in each group (Green: down-regulated genes; Grey: no significant DEGs; Red: Up-regulated gene). (C) Bubble plot of KEGG enrichment of all DEGs. The bar plot: the length represents the number of genes, the abscissa is the ratio of the number of DEGs annotated on the KEGG term to the total number of DEGs, and the ordinate represents the KEGG term. (D) The Real-time PCR analysis of the DEGs mRNA expression in HAE cells.