Natural Product Bruceine A from (L.) Merr. as a Potential LDLR Inhibitor That Facilitates Antiviral Effect

Abstract

Low-density lipoprotein receptor (LDLR), which serves as one of the most major entry receptors for many viruses in both human and mouse cells, plays a vital role in virus infection. However, there are no effective small molecules available to inhibit LDLR expression and exhibit antiviral effects. Here, we screened Bruceine A (BA), a natural product derived from the major constituents in (L.) Merr. which inhibited vesicular stomatitis virus (VSV) infection in vitro. Mechanistically, BA blocked viral adsorption and internalization, facilitating antiviral effects through lysosome-mediated degradation of LDLR. Genetic knockdown of Ldlr exhibited strong antiviral effects. To the best of our knowledge, BA is the first LDLR-selective inhibitor, and our findings reveal that BA may serve as a potent and broad-spectrum virus entry inhibitor based on LDLR entry receptor.

1

Schematic illustration of the antiviral effect of BA. The natural product BA blocks VSV adsorption and internalization to facilitate its antiviral effects through lysosome-mediated degradation of LDLR.

2

Chemical structures of natural products as major constituents in (L.) Merr.

3

Cell viability treated by natural products. The natural products mainly contained quassinoids (A), phenylpropanoids (B), flavonoids (C), and others (D). Cell viability detection of MC38 cells treated with natural products, respectively, at different concentrations for 12 h. Data are represented as means ± SEM (n = 6).

4

BA promoted an antiviral effect in vitro. (A,B) Representative fluorescence images (A) and Western blot (B) of antiviral effects in MC38 infected with VSV virus at an MOI of 1.45 for 1 h, then treated with 0.2 μM and 0.5 μM BA for 11 h, respectively. (C–E) The cell proliferation treated by BA alone (n = 6). EdU assay using MC38 cells treated with 0.5 μM BA for 12 h (C), the percentage of EdU-positive cells (D) and DNA synthesis analysis (E). (F–H) The cell proliferation treated by BA during infection with VSV virus (n = 7). EdU assay using MC38 cells incubated for 1 h in the presence of VSV virus, and then treated with 0.5 μM BA for 11 h respectively (F), the percentage of EdU-positive cells (G), and DNA synthesis analysis (H). Data are presented as mean ± SEM. Unpaired Student’s t-test was performed for statistical analysis and p values at less than 0.05 were considered significant difference. *, p < 0.05; ns = no significance.

5

BA inhibited viral adsorption and internalization in vitro. (A) Short-term infection assays of VSV RNA in MC38 cells treated with 0.2 μM and 0.5 μM BA for the indicated times via qPCR (n = 3). (B) Viral adsorption inhibition assays of BA. MC38 cells pretreated with 0.2 μM and 0.5 μM BA at 4 °C for 1 h, followed by infection with VSV virus for 1 h. The medium was removed, and the cells were washed with ice-cold PBS for three times, then continuously incubated for 5 h. VSV RNA was measured using qPCR (n = 4). (C) Viral internalization inhibition assays of BA, after infection with VSV virus at 4 °C for 1 h, MC38 cells were washed with ice-cold medium for three times and then incubated at 37 °C for 1.5 h to allow for virus internalization in the presence or absence of BA. VSV RNA was measured using qPCR (n = 4). Data are represented as mean ± SEM. Unpaired Student’s t-test was performed for statistical analysis and p values at less than 0.05 were considered significant difference. *, p < 0.05; ****, p < 0.0001.

6

Antiviral effects of natural products. (A–D) Western blot of viral GFP fusion protein in MC38 cells incubated for 1 h in the presence or absence of VSV virus, and then treated with 0.5 μM quassinoids (A), phenylpropanoids (B), flavonoids (C), and others (D) for 11 h, respectively. (E) The schematic illustration of antiviral effects. MC38 cells pretreated, or postinfection treated with BA for the indicated time points. (F) Western blot of viral GFP fusion protein in MC38 cells pretreated or postinfection treated with BA for the indicated time points.

7

Antiviral effects mediated by LDLR. (A) qPCR of Ldlr mRNA in MC38 cells incubated for 1 h in the presence or absence of VSV virus (MOI = 1.45), and then treated with 0.2 μM and 0.5 μM BA for 11 h, respectively (n = 4). (B) Western blot of LDLR in MC38 cells incubated for 1 h in the presence or absence of VSV virus, and then treated with 0.2 μM and 0.5 μM BA for 11 h, respectively. (C–F) Western blot of LDLR and viral GFP fusion protein in MC38 cells incubated for 1 h in the presence or absence of VSV virus, and then treated with 0.5 μM quassinoids (C), phenylpropanoids (D), flavonoids (E), and others (F) for 11 h, respectively. (G,H) The antiviral effects of BA in A549 cells through fluorescence imaging (G) and Western Blotting (H). A549 cells incubated for 1 h in the presence of VSV virus, after then incubated for 11 h in the presence or absence of 0.2 μM BA. (I) MC38 cells were treated with CHX (50 μg/mL), CHX plus MG132 (5 μM), or CHX plus CQ (25 μM) for 12 h in the presence of BA (0.5 μM). (J) The schematic illustration of antiviral effects via siRNA. Representative fluorescence imaging (K) and Western blot (L) of antiviral effects in MC38 cells transfected with siRNA _Ldlr for 48 h and then continuously incubated for 12 h in the presence or absence of VSV virus. Data are presented as mean ± SEM. Unpaired Student’s t-test was performed for statistical analysis and p values at less than 0.05 were considered significant difference. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001.