We previously showed that silymarin inhibits HCV RNA and protein expression in the HCVcc system with JFH-1 virus (6
). demonstrates that in addition to wild type JFH-1 virus, silymarin also blocks replication of HCVcc chimeras, including constructs that contain the H77 (genotype 1a) and J6 (genotype 2a) structural genes in the JFH-1 non-structural gene backbone. Inhibition of HCVcc was 50% for H77/JFH and 75% for J6/JFH chimeras. Thus, silymarin has antiviral actions against multiple HCVcc infectious systems. To determine if silymarin could inhibit binding of HCV virions to cells, we performed virus-cell binding studies at 4°C under conditions where virus binds to but does not enter cells (23
). As shown in , when silymarin was present only during virus-binding, there was little effect on HCV replication. However, if silymarin was added to cells immediately after binding and for the duration of the infection, HCV protein expression was severely impaired. The same effect was observed if silymarin was present during binding and for the duration of the experiment. Next, to determine if silymarin blocked virus entry, we tested the effect of silymarin on viral pseudoparticle entry including HCVpp, VSVpp, and MLVpp. demonstrates that silymarin inhibited the entry of all three pseudotyped viruses. We then examined the effect of silymarin on the fusion of HCVpp with fluorescent liposomes, which examines the effects of compounds on lipid mixing and membrane fusion (24
). As shown in , silymarin drastically inhibited HCVpp-mediated fusion by 80% at 10 μM silymarin, while 20 μM led to a 90% reduction in fusion. DMSO, the solvent control, did not affect fusion. The IC50 of silymarin for membrane fusion inhibition was estimated at 5 μM, far below the doses of silymarin known to confer cytotoxicity in Huh7.5.1 cells (>80 μM, Supplemental Figure S2
, Panel E). The data suggest that silymarin does not affect binding, but inhibits the entry of HCV at the fusion stage.
Antiviral effects of silymarin against HCVcc
Next, we examined the kinetics of inhibition of HCV RNA production. In this experiment we first infected cells for 24 hours, followed by silymarin administration, or IFN-α as a positive control. As shown in , relative to untreated cells, silymarin caused a significant (p<0.01) reduction in JFH-1 RNA production at 48 and 72 hours after treatment. IFN treatment also reduced viral loads. However, significant suppression (p<0.01) of HCV RNA production by IFN started at 18 hours post-treatment and was maintained until 72 hours of treatment. Thus, the kinetics of silymarin mediated suppression of HCV RNA replication were delayed as compared to IFN.
As shown in , silymarin reduced infectious virus yields (measured as FFU/ml) by 5- and 2-fold at 48 and 72 hours post-infection from Huh7.5.1 cells (and in Huh7 cells; data not shown). We can rule out the possibility of carry over silymarin from the initial culture because the supernatants were diluted 1:5–1:1000 before testing on naïve cells. Altogether, the data show that silymarin does not affect virus binding to cells, but inhibits virus entry and fusion, HCV protein and RNA synthesis, and production of progeny viruses in culture supernatants.
Inhibition of HCV RNA and protein expression by silymarin could be due to direct inhibition of viral enzymes, as recently shown for NS5B polymerase activity (25
). Therefore, we tested whether silymarin and silibinin block HCV NS5B polymerase activity. Recombinant NS5B protein from JFH-1 (genotype 2a) lacking the C-terminal 21 amino acids was expressed in E. coli and purified (16
). As shown in , silymarin was able to inhibit JFH-1 NS5B polymerase activity, with an IC50 for silymarin around 300 μM. Silibinin had minimal effects on JFH-1 polymerase, but only at very high doses (IC50 >400μM), which were at least 5–10 fold higher than effective antiviral doses in vitro (6
). At the doses required for inhibition of in-vitro NS5B polymerase activity, silymarin is toxic to cultured Huh7 (6
) and Huh7.5.1 cells (Supplemental Figure S2
Silymarin inhibits JFH-1 polymerase at high dose
We next tested silymarin on RdRp activity of the genotype 1b BK strain and four patient-derived 1b RdRps from patients in the Virahep-C clinical study (26
). The RNA polymerase activities of the patient-derived enzymes were variable (16–104% relative to the well-characterized BK enzyme; ). Silymarin inhibited all five RdRps, with IC50
values ranging from 27.7 to 162 μM. However, in four of the five cases the inhibitory activity of silymarin rapidly plateaued, with maximal inhibition levels of 42.6 to 82.8% relative to the activity in the absence of silymarin (Supplemental Figure S3
). The fifth enzyme (#242) had an inhibition profile that could not be fit to a single-phase exponential decay curve, but its maximal inhibition by silymarin was only 43% and its apparent IC50 was >1,000 μM. Therefore, the IC50 values for most of these subtype 1b RdRps were respectable below the plateau level, but they were poorly inhibited by silymarin at the concentrations employed in the cell culture experiments.
Inhibition Profiles of Genotype 1b RdRps by Silymarin. Polymerase assays were performed and analyzed as described in the Materials and Methods.
If silymarin truly inhibits NS5B polymerase activity, it should be able to inhibit HCV replication in replicon cell lines that do not produce infectious virus. depicts the effects of various doses of silymarin on HCV protein and RNA expression in genotype 1b BB7 subgenomic and FL-NEO genomic replicon cell lines. Silymarin did not significantly inhibit viral protein expression in either cell line when assessed by western blot () or by immunofluorescence (). Silymarin did not inhibit HCV RNA expression in either cell line(). HCV replication was also not inhibited by silymarin in Luc-ubi-neo/ET cells, an independent genotype 1b replicon. (), or in a subgenomic genotype 1a replicon cell line (). In contrast, treatment with IFN-α caused robust suppression of HCV RNA production from the HCV-1a replicon. We tested concentrations of silymarin up to 1000 μM but failed to see any suppression of HCV RNA from the 1a replicon that was independent of cytotoxicity, measured as GAPDH mRNA levels (Supplemental Figure S4
). NS5A protein expression was not affected by silymarin in JFH-1 derived genotype 2a SGR7 () or SGR7.5 replicon cell lines (data not shown). Furthermore, extended treatment of FL-NEO replicon cells (or BB7 cells; data not shown) for 13 days did not affect the levels of HCV NS5A protein (Supplemental Figure S5
). Therefore, silymarin had no antiviral activity against replicon cell lines that did not produce infectious virus. The data in and suggest that silymarin inhibition of NS5B polymerase activity is not a significant component of silymarin's anti-HCV activity in the HCVcc system.
Silymarin does not block HCV replication in HCV replicon cell lines
HCV assembles at lipid droplets (27
), and the virus is thought to exit the infected liver cell by hitching a ride on the apolipoprotein assembly and secretion pathway, in particular MTP-dependent VLDL release (20
). Because silymarin blocked infectious virus production (), we determined whether silymarin also inhibits MTP activity and apoB secretion. In these studies, silymarin was added to cells that were either fully infected (96 hours post-infection) or chronically infected for 14 days. Thus, the experimental design effectively eliminated antiviral effects involving blockade of virus entry and instead allowed us focus on the effects of silymarin on production of progeny viruses. Silymarin inhibited MTP activity in a dose-dependent manner in 14-day chronically infected cells by 25±15% and in non-infected cells by 66±1% at 80 μM (). Naringenin, shown recently to block MTP-dependent virus release (22
), also blocked MTP activity. Silymarin inhibition of MTP activity correlated with reduced apoB secretion in both mock and JFH-1 infected Huh7.5.1 cells (). The small molecule inhibitor of MTP, BMS-200150, served as a positive control for inhibition of apoB secretion. Silymarin inhibition of MTP activity and apoB secretion correlated with a reduction in de novo virion production from fully infected cultures treated for 5 hours (). Importantly, the reduction in infectious virus production was not due to a reduction in intracellular replication, as NS5A protein levels were not affected by the 5-hour treatments with DMSO, silymarin, or BMS-200150 (). Furthermore, the effect on apoB secretion was not unique to Huh7 cells as silymarin also caused dose-dependent suppression of apoB secretion from primary human hepatocytes () and HepG2 cells, as measured by ELISA and western blot (). When we examined intracellular infectious virus as a measure of virus assembly, the general secretion inhibitor Brefeldin A (BFA) caused accumulation of intracellular infectious virus, which was inhibited by the MTP inhibitor BMS-200150, as described (20
). However, silymarin had no effect on BFA-induced accumulation of infectious virus (Supplemental Figure S6
). Collectively, the data demonstrate that silymarin blocks MTP-dependent apoB secretion and infectious virion production into culture supernatants, but does not appear to block virus assembly. We then determined if silymarin blocks other pathways of virus transmission.
It has been recently shown that in addition to releasing virus particles into culture medium, HCV is capable of direct cell-to-cell transmission (21
). To examine effects of silymarin on this antibody insensitive route of transmission, we used a novel assay where fluorescently labeled infected producer cells were mixed with unlabeled naïve cells and HCV NS5A protein expression was detected using antibodies labeled in the red spectrum. Silymarin reduced both total and cell-to-cell transmission (). We also observed equal suppression of both total and cell-to-cell transmission (), suggesting that silymarin does not discriminate between routes of virus transmission.
Silymarin inhibits total virus transmission and cell-to-cell spread