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Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL, however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites.
Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.
Worldwide about 170 million people are chronically infected with HCV, a positive-strand RNA virus belonging to the Flaviviridae family and leading to severe liver disease in many cases. Only 50% of the patients respond to therapy with pegylated IFNα (pegIFNα) + Ribavirin (RBV) for yet unknown reasons. The newly available direct acting antivirals (DAA) telaprevir and boceprevir enhance sustained viral response rates up to 70–75% during triple therapy (pegIFNα + RBV + DAA) of genotype 1 infected patients, but are accompanied by numerous and potentially severe adverse effects (1). Therefore there is still an urgent need for more potent and better tolerated therapeutic options.
Silymarin is an extract from the milk thistle plant (Silybum marianum) and contains a mixture of several compounds with silibinin (SbN) being the major component consisting of the two flavonolignans silybin A and silybin B. In contrast to the oral formulation (2), succinylated silibinin (Legalon-SIL® (SIL)), which is administered intravenously, has antiviral effects in chronic HCV patients. This mixture has been primarily used to prevent re-infection of the graft after liver transplantation (3–5) and for the treatment of IFNα/RBV nonresponders (6–9). Importantly, several individual case reports showed that patients could even be cured from HCV by SIL monotherapy with few adverse side effects, underlining the antiviral potency of this therapeutic (3–5). However, the mode of action (MOA) of SIL is currently under debate. Ahmed-Belkacem et al. (10) suggested that SIL targets viral RNA replication by direct inhibition of the HCV RNA dependent RNA polymerase (RdRp). Wagoner et al. (11) observed an efficient block of viral RNA replication of HCV genotype 1b (gt1b) replicons by SIL as well, accompanied by additional effects of SIL on viral entry as well as progeny virus particle production at very high SIL concentrations. In this study we aimed to clarify the MOA of SIL by analyzing the effect of SIL on the replication of different viral genotypes as well as emergence of SIL-resistant viruses in vitro and in vivo.
Legalon SIL® (SIL) (lyophilized, Madaus, Cologne) was resuspended to 28.5mg/ml (corresponding to 67.6mM) in sterile water and stored at −70°C. Further dilution was carried out in complete DMEM.
All amino acid and nucleotide numbers refer to the position of the corresponding amino acid in the complete HCV genomes of JFH1, Con1 and H77 (GenBank accession no. AB047639, AJ238799 and AF011751, respectively). Detailed information can be found in Supplementary Materials and Methods.
SIL was diluted in complete DMEM to concentrations ranging from 1μM to 1mM. In assays based on transient transfection of replicon RNA or stable replicon cells SIL treatment occurred 4h after seeding of the cells until cell lysis at 48h. In the infection assay with JcR-2a the drug was added together with the virus. At 6h post infection, medium was replaced with fresh medium containing SIL and incubated for 48h. In transfection assays for immunofluorescence or electron microscopic analyses SIL was added immediately after transfection until fixation 24h later.
HCV genomes were amplified in two fragments by RT-nested PCR from total RNA purified from the serum of two patients before and after SIL therapy. Sequence data of both patients were deposited in GenBank with following IDs: JQ914271 - patient A before SIL treatment, JQ914272 - patient A after SIL treatment, KC155254 - patient A 21 months after cessation of SIL therapy, JQ914273 - patient B before SIL treatment, JQ914274 - patient B after SIL treatment. Primer sequences and protocols for RNA purification and RT-PCR are given in Supplementary Materials and Methods. Written informed consent was obtained from both patients. No donor organs were obtained from executed prisoners or other institutionalized persons.
To elucidate the MOA of SIL, we defined which part of the HCV lifecycle was primarily inhibited by the drug. We first determined the SIL sensitivity of persistent subgenomic reporter replicons of genotype 1b (Luc/neo Con1) and 2a (Luc/neo JFH1) mimicking chronic HCV infection (Fig. 1A, B). Luc/neo Con1 replication was strongly inhibited by SIL, whereas Luc/neo JFH1 replication was not affected (Fig. 1B), in line with recently published data (11) and in absence of cytostatic effects of the drug (Fig. 1D). Similar results were obtained upon transient transfection of luciferase replicons of the same isolates (Fig. 1A, C). In contrast and in agreement with previous reports, Silibinin (SbN) had no specific impact on HCV RNA replication of Con1 or JFH1 ((11); Fig. S1). Since replicons based on isolate JFH1 replicated much more efficiently in cell culture compared to Con1 (12) we tested SIL sensitivity of JFH1 replicons harboring a chimeric 5′UTR or/and X-tail, replicating with comparable efficiency (5′UTR or X-tail Con1) or even less efficiently (5′UTR/X-tail Con1) than Con1 (Fig. 1E, (12). However, none of the replicons bearing the JFH1 nonstructural protein coding sequence were inhibited by SIL treatment (Fig. 1F), suggesting that JFH1 RNA replication is intrinsically unresponsive to SIL treatment. We also found no significant impact of SIL on infection of a chimeric reporter virus (JcR-2a, Fig. 1A, C (13)), arguing against a substantial inhibition of HCV gt2a entry and replication by SIL. The data demonstrate a strong inhibition of persistent and transient HCV genotype 1b RNA replication by SIL and little impact on replication or infection of genotype 2a in cell culture.
We next selected gt1b replicon cells for resistance to SIL in vitro and obtained a few cell clones with persistent HCV replication in presence of SIL. Sequence analysis of RT-PCR amplicons identified seven mutations (Fig. 2A), which were conserved in at least one of eight SIL resistant replicon cell clone (Table S1). All mutations were introduced individually into a LucCon1 replicon (Fig. 1A) and analyzed for phenotypic effects on SIL sensitivity and replication fitness. Only Q1914R in the C-terminal region of NS4B, present in three SIL resistant replicon clones, significantly reduced HCV sensitivity to SIL (ca. 2fold IC90; Fig. 2B, C; Table 1). Q1914R furthermore impaired replication fitness (Fig. 2D, E), concordant with the high conservation of glutamine at this position across all HCV genotypes, including isolate JFH1 (14).
Collectively, the data indicate that SIL-resistant replicon clones were selected in vitro and a mutation located in the C-terminal region of NS4B (Q1914R) conferred HCV gt1b resistance to SIL.
Since HCV resistance to SIL could be selected for in vitro, we asked whether SIL resistance occurred in vivo. We focused on a male patient with chronic HCV genotype 1a infection previously treated unsuccessfully with IFNα monotherapy, IFNα/RBV and pegIFNα/RBV. The patient underwent extended right lobe liver transplantation and during the anhepatic phase SIL therapy was initiated with 20mg/kg body weight/day based on previous reports (4;8;15). After liver transplantation, viral titers dropped rapidly followed by a continuous decrease of viral load until day 13 after onset of treatment (Fig. 3A). From day 18 to 27 viral load increased back to basal level (viral breakthrough) suggesting the emergence of resistance to SIL. To identify mutations potentially conferring SIL resistance, we amplified and directly sequenced the viral genome before and after viral breakthrough in patient A (Fig. 3B). Before SIL treatment we observed several polymorphisms (Fig. 3B, grey lines) and a subpopulation of viral quasispecies carrying a deletion of the coding sequence of E2 and parts of E1 and p7 (nt 1204 to 2639) (Fig. 3C). After treatment the viral sequence lacked polymorphisms and the subgenome was no longer detectable supporting the concept that the viral quasispecies had passed a genetic bottleneck followed by selection of SIL resistant variants. However, seven novel and conserved mutations exclusively within the nonstructural proteins emerged: G963S (NS2), P/T1112S (NS3), F1809L, D1939N, T1946A (NS4B), E2265D (NS5A) and V2431I (NS5B) (Fig. 3B, black lines, Table S2).
In summary, we analyzed a patient with viral breakthrough under SIL treatment, revealing seven conserved mutations potentially contributing to SIL resistance.
To characterize phenotypic effects of mutations identified in patient A, we used a highly cell culture adapted variant of the HCV genotype 1a isolate H77, termed H77S (16) (Fig. 4A). Replication fitness and SIL sensitivity of the H77S reporter replicon was similar to LucCon1 (Fig. 4D, 4B, Table 2). We chose F1809L, D1939N and E2265D for further phenotypic analysis due to their high degree of conservation or because they matched to the SIL resistance profile of HCV isolates in cell culture (Table S2). G963S was excluded since NS2 is not required for RNA replication (17). T1946A was already present in H77S, whereas V2431I was found in the SIL sensitive isolate Con1 (Table S2). Mutations chosen for phenotypic analysis of SIL resistance were introduced individually and in combinations into the H77S reporter replicon (Fig. 4A) and analyzed for SIL sensitivity (Fig. 4B, C; Table 2) and replication fitness (Fig. 4D, E). Mutation D1939N located in the C-terminal region of NS4B significantly reduced SIL sensitivity of H77S (ca. 1.7fold IC90, Fig. 4C; Table 2), accompanied by a significant impairment of replication fitness (Fig. 4E). F1809L slightly reduced SIL sensitivity but significantly increased replication fitness (Fig. 4E). Interestingly, double mutant F1809L+D1939N was less sensitive to SIL than the single mutants (ca. 2.5fold IC90; Fig. 4C, Table 2), arguing for a contribution of both NS4B mutations to SIL resistance in the examined patient. Replication fitness was furthermore restored in the double mutant F1809L+D1939N compared to the D1939N single mutant (Fig. 4E) suggesting that the F1809L mutation compensated for the fitness cost associated with D1939N. NS5A mutation E2265D had no impact on the resistance to SIL, neither individually nor in combination (Fig. 4C, Table 2). The involvement of F1809L+D1939N in SIL-resistance was supported by sequence analysis of viral genomes 21 months after cessation of SIL therapy, since both positions were found reverted, in contrast to T1946A, E2265D and V2431I (Fig. 3D). Additional reversion were found at positions 1112 in NS3 and 963 in NS2. However, mutation P1112S did not contribute to SIL resistance in vitro, neither as a single mutation introduced in the H77S replicon, nor in combination with F1809L+D1939N (data not shown). Cumulatively, phenotypic analysis of mutations identified in vivo supported a mechanism of action of SIL involving NS4B.
NS4B is primarily involved in inducing membrane alterations known as the membranous web which harbors the viral replication complex (18). Previous work suggests NS4B having a complex membrane topology (Fig. 5A, reviewed in (14)). While position 1809 is located in a proposed short ER-luminal loop between predicted transmembrane segments 1 and 2 (TM1 and 2), both major mutations conferring SIL resistance mapped to the C-terminal region of NS4B. Interestingly, substitutions Q1914R and D1939N are located in a region of amphipathic α-helices which are proposed to be embedded in the membrane interface and to point to the cytosol, thereby providing a potential platform for protein-protein interactions (Fig. 5A,B, (19)). Indeed, recent studies have delineated the functional importance of the NS4B C-terminal mediated by homo- and heterotypic interactions between N- and C-termini of NS4B (19–21). We employed a fluorescence resonance energy transfer (FRET)-based assay described earlier (21) to address whether SIL affected self-interaction of NS4B (Fig. 5C). However, SIL had no effect on FRET efficiency, suggesting that SIL did not interfere with NS4B oligomerization. Interactions of NS4B with other HCV nonstructural proteins have been reported as well (reviewed in (14)). We therefore generated chimeric replicons based on the SIL resistant JFH1 isolate, replacing parts of the nonstructural protein coding region by homologous sequences of the SIL sensitive isolate Con1 (Fig. 5D), to analyze which parts of the coding region transferred SIL sensitivity. Exclusive exchange of the NS4B sequence of JFH1 significantly increased SIL sensitivity of the resulting chimera compared to JFH1, but sensitivity did not reach the level of Con1 (NS4B Con1, Fig. 5E,F). We then added the NS3/4A and/or the NS5A coding sequences of Con1 (Fig. 5D). The construct harboring the NS4B and NS5A sequence of Con1 was not replication competent, as well as a replicon containing only NS3/4A of Con1 (NS4B-5A Con1, NS3/4A Con1, respectively; Fig. S3). However, SIL sensitivity of the chimeric replicons NS3-4B Con1 and NS3-5A Con1 was identical to Con1 (Fig. 5D–F), suggesting that NS3/4A is an additional important determinant of SIL response and arguing against a major contribution of NS5A.
NS4B as well as NS3 have been described to induce membrane alterations (22) and the interaction between both proteins might critically contribute to the formation of functional HCV replication sites. These correspond to accumulations of vesicular structures (18), mainly composed of double membrane vesicles (DMVs) (13). To address a potential interference of SIL on the biogenesis of virus induced membrane alterations we transiently expressed NS3-5B from Con1 or JFH1 in Huh7-T7 cells and analyzed the impact of SIL treatment on the membranous web. SIL treatment had no impact on the distribution of NS4B, NS3 and NS5A and did not change the degree of colocalization of these proteins (Fig. S4 and S5). We also found no alterations in intracellular PI4P levels, suggesting that SIL does not affect the activation of PI4KIIIα, an essential host factor of HCV replication ((13), Fig. S6). However, ultrastructural examination revealed that in cells expressing NS3-5B of the Con1 isolate and treated with SIL, most of the vesicles observed were multi-membrane vesicles (MMVs) and no longer DMVs (Fig. 6A,B), suggesting that SIL indeed modulated the morphology of viral replication sites. In contrast, SbN had no impact on the proportion of MMVs, confirming the specificity of the changes caused by SIL (Fig. S8). Importantly, the morphology of membrane alterations induced by the SIL resistant genotype 1b mutant Q1914R and by the resistant JFH1 isolate was not affected by SIL, supporting the assumption that SIL indeed acts by disturbing viral replication sites (Fig. 6A,C and Fig. S7).
Collectively these data suggest that SIL treatment affects the morphogenesis of viral replication sites by targeting NS4B, probably by modulating a critical interaction with NS3/4A.
The mechanisms by which SIL inhibits HCV infection in vivo are currently under debate. Previous studies observed a direct inhibition of the viral polymerase in vitro by SIL and SbN (10;11;23), suggestive for the mechanism in targeting RNA replication. However, our data confirm that inhibition of viral RNA replication indeed is the primary MOA of SIL, but implicate that NS4B is a candidate target: First we identified in vitro and in vivo mutations in NS4B conferring partial SIL resistance. Second, SIL modulated the ultrastructure of genotype 1b membrane alterations induced by the viral nonstructural proteins, which are mainly mediated by NS4B (22), but not those generated by gt2a or by a SIL resistant gt1b variant. Third, the NS4B and NS3/4A coding sequence of SIL sensitive isolate Con1 rendered resistant JFH1 fully sensitive to SIL. Taken together, these results suggest that the antiviral activity of SIL is at least in part mediated by NS4B, probably by targeting an interaction with NS3/4A. A critical interaction between NS3 and NS4B has already been suggested by a previous study, based on genetic evidence (24). The fact that we identified mutations conferring SIL resistance only in NS4B might simply reflect restrictions of the isolates included in this study and clearly does not exclude potential alternative sites in NS3 conferring resistance to SIL in other HCV isolates.
NS4B is the key factor inducing membrane alterations harboring the HCV replication sites, which mainly consist of DMVs and to a lesser extend MMVs (13;20). The biogenesis of these vesicular structures is poorly defined, but they are believed to be generated by a concerted action of the nonstructural proteins, with NS4B as the main actor (22). The fact that SIL strongly reduced the number of DMVs and triggered the formation of MMVs therefore argues for an interference with the morphogenesis of viral replication sites. Mutations conferring resistance were mainly located at the C-terminal amphipathic α-helices (Fig. 5A), and affected conserved residues facing the cytosol (Fig. 5B). Those sites could provide a platform for the interaction with other NS-proteins, like NS3/4A or with host factors. Although our studies suggest that SIL might affect a critical NS4B interaction with NS3/4A, we cannot exclude that SIL in addition impacts on interactions of other NS proteins or host factors. The membrane activity reported for Silibinin (25) might also contribute to the antiviral action of SIL, due to the tight membrane association of all viral proteins. The precise mechanism of action of SIL remains to be determined. However, SIL is not the first drug reported to target NS4B (reviewed in (26)), but resistance profiles and suggested MOAs of other classes of NS4B inhibitors suggest that SIL represents a novel type with a unique mode of action.
In contrast to other studies (10;11), we found no evidence for a substantial inhibition of genotype 2a HCVcc infection by SIL. Still, we might have missed effects on entry due to differences in the experimental design. The fact that mutation G963S at a highly conserved position in NS2 was found after therapy but reverted back later suggests that NS2 might be a target of SIL too. Such additional MOAs complementing inhibition of RNA replication might also explain cure or at least absence of viral breakthrough in SIL monotherapy (3;4;27;28) due to a higher barrier to resistance.
The limited data on SIL therapy outcomes include sustained virological response (3–7), initial suppression of viral replication followed by rebound (relapse or breakthrough, patient A, (5;6)) and non-response (patient B, Fig. S2 (6;15)). Rutter et al. suggested low viral load as the most valuable predictor of treatment response in their study (6). This might account for successful therapies post LTx (3;4), since a small pool of virus variants at the onset of therapy limits the chance to select for resistance. The main determinant for successful SIL monotherapy after LTx might therefore be the barrier to resistance of the HCV quasispecies in a patient. In the case of patient A, two mutations in highly conserved residues in NS4B seemed to be sufficient for viral breakthrough; in case of other isolates, more mutations might be required or the fitness costs associated with these mutations might be higher. Our data furthermore indicate that SIL might not be effective in genotype 2 since we found no inhibition of RNA replication of genotype 2a isolate JFH1. Interestingly, this is corroborated by two clinical reports about nonresponse of genotype 2 patients to SIL ((29) and S. Beinhardt, personal communication). However, larger patient cohorts will be required to clarify determinants of treatment failure, particularly since a second gt1a patient treated with SIL (patient B) did not respond to SIL therapy, without having obvious alterations in the NS4B sequence (Fig. S2)
In conclusion, our data indicate that SIL is an efficient inhibitor of HCV RNA replication at least in part by targeting NS4B. The emergence of resistance in vivo and in vitro suggests that SIL should not be used in monotherapy. Although it seems unlikely that SIL will become a major drug in HCV therapy, it represents a promising component of future combination therapies particularly in difficult-to-treat patient cohorts, due to its novel mode of action and unique resistance profile.
This project was funded by grants from the Deutsche Forschungsgemeinschaft (FOR 1202, TP1 and TP3 to R.B. and V.L., respectively, and LO 1556/1-2 to V.L.), the Swiss National Science Foundation (31003A-138484 to D.M.), and the french ANRS (to F.P.). S.J.P. is partially supported by NIH grants U19AI066328, R01AT006842, and R56AI091840.
We thank Jens Bukh for plasmid pCV-H77c, Takaji Wakita for isolate JFH1, Charlie Rice for Huh7.5 cells, antibody 9E10 and helpful discussions and Simone Hoppe and Ulrike Herian for excellent technical assistance.
The authors have no conflict of interest.