Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro

Abstract

Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro.

Fig 1. The molecular structure of luteoloside and its effects on RD cell viability.

(A) The molecular structure of luteoloside. (B) Luteoloside was diluted as various concentrations as indicated in triplicate. The cytotoxicity of the compound was determined by MTS assay after drug-incubation for 48 h. The viability of cells upon DMEM medium without luteoloside (0 mM) was set as 100%. Data shown are the means ± SE from 6 independent measurements (n = 6). Asterisk meant the data differed from the blank (0 mM) significantly at P<0.05 level according to t-test.

Fig 2. The antiviral effects of luteoloside against EV71 in RD cells.

RD cells were infected 0.3 MOI (A) or 1.0 MOI (B) EV71 with or without different concentrations of luteoloside are shown. Uninfected cells were taken as control group. The cell viability was detected using MTS cell proliferation assay kit at 48 hpi. The viability of control group was set as 100%. Data shown are the means ± SE from 6 independent measurements (n = 6). Asterisk meant the data differed from the EV71 group significantly at P<0.05 level according to t-test.

Fig 3. Luteoloside inhibited EV71 production in a time-dependent manner.

RD cells were infected EV71 (MOI = 0.3). 0.5 mM luteoloside were added at the indicated time points, respectively. Uninfected cells were used as a control group. The cell viability was detected using MTS cell proliferation assay kit at 48 hpi. The viability of control group was set as 100%. Data shown are the means ± SE from 6 independent measurements (n = 6), asterisk means P<0.05 according to t-test comparing with EV71 group.

Fig 4. Luteoloside reduced EV71 production.

(A) RD cells grown in 6-well-plate were infected with EV71 (MOI = 0.3) in the presence or absence of 0.5 mM luteoloside. The intercellular and extracellular virions were collected at the indicated time points for plaque reduction assay by freeze-thawing. (B) RD cells were infected with EV71 (MOI = 0.3). 0.5 mM luteoloside were supplemented to infected RD cells according to different protocols (C) respectively. The cell viability was detected using MTS cell proliferation assay kit at 48 hpi. The viability of control group was set as 100%. Data shown are the means ± SE from 6 independent measurements (n = 6). Asterisk meant the data differed from the EV71 group significantly at P<0.05 level according to t-test.

Fig 5. Luteoloside inhibited the production of viral proteins and nuclear acids.

RD cells grown in 6-well-plate were infected with EV71 (MOI = 0.3) in the presence or absence of 0.5 mM luteoloside. Cells were lysed for total RNA and protein extraction at 12 hpi, respectively. (A) The RNA load was determined using the real-time PCR kit specific to VP1 gene. Data shown are the means ± SE from 6 independent measurements (n = 6). Asterisk meant the data differed from the EV71 group significantly at P<0.05 level according to t-test. (B) Protein samples, unified to 30 μg, were subjected to 12.5% SDS-PAGE and then transferred to PVDF membrane to detect the level of EV71 VP1 protein. The amount of β-tubulin was used as internal standard. VP1 band intensity was analyzed and normalized to corresponding band intensity of β-tubulin. The band intensity of EV71 group was set as 100%. Data shown are the means ± SE from 3 independent measurements (n = 3).

Fig 6. Dose-dependent inhibitory of luteoloside on EV71 3C protease activity in vitro.

Two-fold dilutions of luteoloside were added in 3C protease analysis system. The enzymatic activity of HRP exposed to various treatments was calculated. Blank (0 mM luteoloside) and 0.5 mM rutin were set as negative and positive control, respectively. Data shown are the means ± SE values of 3 independent experiments (n = 3).