Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV''s NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.     

Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V_HH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V_HH from a humanized-camel VH/V_HH display library. VH/V_HH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V_HH hallmark and were designated V_HH6 and V_HH24; other clones were conventional VH, designated VH9 and VH13. All VH/V_HH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V_HH6 and V_HH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V_HH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.     

Anti-hepatitis C virus activity of tamoxifen reveals the functional association of estrogen receptor with viral RNA polymerase NS5B

Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. HCV genome replication occurs in the replication complex (RC) around the endoplasmic reticulum membrane. However, the mechanisms regulating the HCV RC remain widely unknown. Here, we used a chemical biology approach to show that estrogen receptor (ESR) is functionally associated with HCV replication. We found that tamoxifen suppressed HCV genome replication. Part of ESRalpha resided on the endoplasmic reticulum membranes and interacted with HCV RNA polymerase NS5B. RNA interference-mediated knockdown of endogenous ESRalpha reduced HCV replication. Mechanistic analysis suggested that ESRalpha promoted NS5B association with the RC and that tamoxifen abrogated NS5B-RC association. Thus, ESRalpha regulated the presence of NS5B in the RC and stimulated HCV replication. Moreover, the ability of ESRalpha to regulate NS5B was suggested to serve as a potential novel target for anti-HCV therapeutics.     

Non-nucleoside inhibitors binding to hepatitis C virus NS5B polymerase reveal a novel mechanism of inhibition

The RNA-dependent RNA polymerase (NS5B) from hepatitis C virus (HCV) is a key enzyme in HCV replication. NS5B is a major target for the development of antiviral compounds directed against HCV. Here we present the structures of three thiophene-based non-nucleoside inhibitors (NNIs) bound non-covalently to NS5B. Each of the inhibitors binds to NS5B non-competitively to a common binding site in the "thumb" domain that is approximately 35 Angstroms from the polymerase active site located in the "palm" domain. The three compounds exhibit IC(50) values in the range of 270 nM to 307 nM and have common binding features that result in relatively large conformational changes of residues that interact directly with the inhibitors as well as for other residues adjacent to the binding site. Detailed comparisons of the unbound NS5B structure with those having the bound inhibitors present show that residues Pro495 to Arg505 (the N terminus of the "T" helix) exhibit some of the largest changes. It has been reported that Pro495, Pro496, Val499 and Arg503 are part of the guanosine triphosphate (GTP) specific allosteric binding site located in close proximity to our binding site. It has also been reported that the introduction of mutations to key residues in this region (i.e. Val499Gly) ablate in vivo sub-genomic HCV RNA replication. The details of NS5B polymerase/inhibitor binding interactions coupled with the observed induced conformational changes provide new insights into the design of novel NNIs of HCV.     

SAR study of bisamides as cyclophilin a inhibitors for the development of host-targeting therapy for hepatitis C virus infection

The therapy of chronic hepatitis C virus infections has significantly improved with the development of direct-acting antivirals (DAAs), which contain NS3/4A protease, NS5A, and NS5B polymerase inhibitors. However, mutations in specific residues in these viral target genes are associated with resistance to the DAAs. Especially inhibitors of NS3/4A protease and NS5A, such as grazoprevir and velpatasvir, have a low barrier to resistant mutations. As a result, the mutations influence the virological outcomes after DAA treatment. CypA inhibitors, as host-targeted agents, act on host factors to inhibit HCV replication, exhibiting a high resistance barrier and pan-genotype activities against HCV. Therefore, they can be developed into alternative, more effective anti-HCV agents. However, CypA inhibitors are natural products and analogs. Based on previous studies, bisamide derivatives were designed and synthesized to develop a novel class of CypA inhibitors. Bisamide derivative 7c is a promising compound with potent anti-HCV activity at subtoxic concentrations. Surface plasmon resonance experiments revealed that 7c directly binds to CypA. All these studies indicated that the derivative 7c is a potent CypA inhibitor, which can be used as a host-targeted agent in combination with other antiviral agents for anti-HCV treatment.     

Crystallographic identification of a noncompetitive inhibitor binding site on the hepatitis C virus NS5B RNA polymerase enzyme

The virus-encoded nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase and is absolutely required for replication of the virus. NS5B exhibits significant differences from cellular polymerases and therefore has become an attractive target for anti-HCV therapy. Using a high-throughput screen, we discovered a novel NS5B inhibitor that binds to the enzyme noncompetitively with respect to nucleotide substrates. Here we report the crystal structure of NS5B complexed with this small molecule inhibitor. Unexpectedly, the inhibitor is bound within a narrow cleft on the protein's surface in the "thumb" domain, about 30 A from the enzyme's catalytic center. The interaction between this inhibitor and NS5B occurs without dramatic changes to the structure of the protein, and sequence analysis suggests that the binding site is conserved across known HCV genotypes. Possible mechanisms of inhibition include perturbation of protein dynamics, interference with RNA binding, and disruption of enzyme oligomerization.     

Network based analysis of hepatitis C virus core and NS4B protein interactions

Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. Here we attempt to further our understanding of the biological context of protein interactions in HCV pathogenesis, by investigating interactions between HCV proteins Core and NS4B and human host proteins. Using the yeast two-hybrid (Y2H) membrane protein system, eleven human host proteins interacting with Core and 45 interacting with NS4B were identified, most of which are novel. These interactions were used to infer overall protein interaction maps linking the viral proteins with components of the host cellular networks. Core and NS4B proteins contribute to highly compact interaction networks that may enable the virus to respond rapidly to host physiological responses to HCV infection. Analysis of the interaction networks highlighted enriched biological pathways likely influenced in HCV infection. Inspection of individual interactions offered further insights into the possible mechanisms that permit HCV to evade the host immune response and appropriate host metabolic machinery. Follow-up cellular assays with cell lines infected with HCV genotype 1b and 2a strains validated Core interacting proteins ENO1 and SLC25A5 and host protein PXN as novel regulators of HCV replication and viral production. ENO1 siRNA knockdown was found to inhibit HCV replication in both the HCV genotypes and viral RNA release in genotype 2a. PXN siRNA inhibition was observed to inhibit replication specifically in genotype 1b but not in genotype 2a, while SLC25A5 siRNA facilitated a minor increase in the viral RNA release in genotype 2a. Thus, our analysis can provide potential targets for more effective anti-HCV therapeutic intervention.     

A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon’s anti-HCV action and for screening and testing new anti-HCV drugs.     

Comparative analysis of intracellular inhibition of hepatitis C virus replication by small interfering RNAs

Several synthetic siRNAs were designed to target various regions of hepatitis C virus (HCV) replicon RNA. The antiviral efficacies of the siRNAs were compared using real time PCR and western blot assessment. siRNAs targeting either specific coding region of HCV NS3 or NS5B were the most efficacious in terms of gene silencing and inhibitory activity of the HCV replicon replication. There was no activation of genes involved in innate immune response by the HCV-specific siRNA, indicating that HCV replication inhibition was not due to non-specific antiviral response. Moreover, 5′-RACE PCR analysis showed that the silencing effect by the siRNAs was mainly caused by specific cleavage of targeted HCV genomic RNA. These findings suggest that RNAi targeting HCV coding regions could provide a useful approach to anti-HCV treatment.     

Hepatitis C Virus NS5B and Host Cyclophilin A Share a Common Binding Site on NS5A

Nonstructural protein 5B (NS5B) is essential for hepatitis C virus (HCV) replication as it carries the viral RNA-dependent RNA polymerase enzymatic activity. HCV replication occurs in a membrane-associated multiprotein complex in which HCV NS5A and host cyclophilin A (CypA) have been shown to be present together with the viral polymerase. We used NMR spectroscopy to perform a per residue level characterization of the molecular interactions between the unfolded domains 2 and 3 of NS5A (NS5A-D2 and NS5A-D3), CypA, and NS5B_Δ21. We show that three regions of NS5A-D2 (residues 250–262 (region A), 274–287 (region B), and 306–333 (region C)) interact with NS5B_Δ21, whereas NS5A-D3 does not. We show that both NS5B_Δ21 and CypA share a common binding site on NS5A that contains residues Pro-306 to Glu-323. No direct molecular interaction has been detected by NMR spectroscopy between HCV NS5B_Δ21 and host CypA. We show that cyclosporine A added to a sample containing NS5B_Δ21, NS5A-D2, and CypA specifically inhibits the interaction between CypA and NS5A-D2 without altering the one between NS5A-D2 and NS5B_Δ21. A high quality heteronuclear NMR spectrum of HCV NS5B_Δ21 has been obtained and was used to characterize the binding site on the polymerase of NS5A-D2. Moreover these data highlight the potential of using NMR of NS5B_Δ21 as a powerful tool to characterize in solution the interactions of the HCV polymerase with all kinds of molecules (proteins, inhibitors, RNA). This work brings new insights into the comprehension of the molecular interplay between NS5B, NS5A, and CypA, three essentials proteins for HCV replication.     

Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.     

Interaction networks of hepatitis C virus NS4B: implications for antiviral therapy

Hepatitis C virus (HCV) is an important human pathogen infecting more than 170 million people worldwide with approximately three million new cases each year. HCV depends heavily on interactions between viral proteins and host factors for its survival and propagation. Among HCV viral proteins, the HCV non-structural protein 4B (NS4B) has been shown to mediate virus-host interactions that are essential for HCV replication and pathogenesis and emerged as the target for anti-HCV therapy. Here, we reviewed recent knowledge about the NS4B interaction networks with host factors and its possible regulatory mechanisms, which will both advance our understanding of the role of NS4B in HCV life cycle and illuminate potential viral and host therapeutic targets.     

Direct interaction between alpha-actinin and hepatitis C virus NS5B

It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication. By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library. For one of these, alpha-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis. Experiments with deletion mutants showed that domains NS5B(84-95), NS5B(466-478), and alpha-actinin(621-733) are responsible for the interaction. Studies of the HCV subgenomic replicon system with small interference RNA indicate that alpha-actinin is essential for HCV RNA replication. Our results suggest alpha-actinin may be a component of the HCV replication complex.     

VAPC, an human endogenous inhibitor for hepatitis C virus (HCV) infection, is intrinsically unstructured but forms a "fuzzy complex" with HCV NS5B

Nearly 200 million people are infected by hepatitis C virus (HCV) worldwide. For replicating the HCV genome, the membrane-associated machinery needs to be formed by both HCV non-structural proteins (including NS5B) and human host factors such as VAPB. Recently, the 99-residue VAPC, a splicing variant of VAPB, was demonstrated to inhibit HCV replication via binding to NS5B, thus acting as an endogenous inhibitor of HCV infection. So far, the structure of VAPC remains unknown, and its interaction with NS5B has not been biophysically characterized. In this study, we conducted extensive CD and NMR investigations on VAPC which led to several striking findings: 1) although the N-terminal 70 residues are identical in VAPC and VAPB, they constitute the characteristic β-barrel MSP fold in VAPB, while VAPC is entirely unstructured in solution, only with helical-like conformations weakly populated. 2) VAPC is indeed capable of binding to NS5B, with an average dissociation constant (Kd) of ～20 μM. Intriguingly, VAPC remains dynamic even in the complex, suggesting that the VAPC-NS5B is a "fuzzy complex". 3) NMR mapping revealed that the major binding region for NS5B is located over the C-terminal half of VAPC, which is composed of three discrete clusters, of which only the first contains the region identical in VAPC and VAPB. The second region containing ～12 residues appears to play a key role in binding since mutation of 4 residues within this region leads to almost complete loss of the binding activity. 4) A 14-residue mimetic, VAPC-14 containing the second region, only has a ～3-fold reduction of the affinity. Our study not only provides critical insights into how a human factor mediates the formation of the HCV replication machinery, but also leads to design of VAPC-14 which may be further used to explore the function of VAPC and to develop anti-HCV molecules.     

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.     

A comprehensive structure-function comparison of hepatitis C virus strain JFH1 and J6 polymerases reveals a key residue stimulating replication in cell culture across genotypes

The hepatitis C virus (HCV) genotype 2a isolate JFH1 represents the only cloned HCV wild-type sequence capable of efficient replication in cell culture as well as in vivo. Previous reports have pointed to NS5B, the viral RNA-dependent RNA polymerase (RdRp), as a major determinant for efficient replication of this isolate. To understand the contribution of the JFH1 NS5B gene at the molecular level, we aimed at conferring JFH1 properties to NS5B from the closely related J6 isolate. We created intragenotypic chimeras in the NS5B regions of JFH1 and J6 and compared replication efficiency in cell culture and RdRp activity of the purified proteins in vitro, revealing more than three independent mechanisms conferring the role of JFH1 NS5B in efficient RNA replication. Most critical was residue I405 in the thumb domain of the polymerase, which strongly stimulated replication in cell culture by enhancing overall de novo RNA synthesis. A structural comparison of JFH1 and J6 at high resolution indicated a clear correlation of a closed-thumb conformation of the RdRp and the efficiency of the enzyme at de novo RNA synthesis, in accordance with the proposal that I405 enhances de novo initiation. In addition, we identified several residues enhancing replication independent of RdRp activity in vitro. The functional properties of JFH1 NS5B could be restored by a few single-nucleotide substitutions to the J6 isolate. Finally, we were able to enhance the replication efficiency of a genotype 1b isolate with the I405 mutation, indicating that this mechanism of action is conserved across genotypes.     

Pyridobenzothiazole derivatives as new chemotype targeting the HCV NS5B polymerase

Hepatitis C virus (HCV) infection has been recognized as the major cause of liver failure that can lead to hepatocellular carcinoma. Among all the HCV proteins, NS5B polymerase represents a leading target for drug discovery strategies. Herein, we describe our initial research efforts towards the identification of new chemotypes as allosteric NS5B inhibitors. In particular, the design, synthesis, in vitro anti-NS5B and in cellulo anti-HCV evaluation of a series of 1-oxo-1H-pyrido[2,1-b][1,3]benzothiazole-4-carboxylate derivatives are reported. Some of the newly synthesized compounds showed an IC(50) ranging from 11 to 23 μM, and molecular modeling and biochemical studies suggested that the thumb domain could be the target site for this new class of NS5B inhibitors.     

Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.     

Hepatitis C virus nonstructural protein 5B is involved in virus morphogenesis

The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.