A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development

Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs

Conserved GXXXG- and S/T-Like Motifs in the Transmembrane Domains of NS4B Protein Are Required for Hepatitis C Virus Replication▿

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle