HCV NS5A is a multifunctional protein involved in viral replication and pathogenesis (29
). In a previous study, we have shown that NS5A specifically interacts with FKBP8 and recruits Hsp90 to the viral RNA replication complex through the interaction of the carboxylate clump structure of FKBP8 with the C-terminal MEEVD motif of Hsp90 (37
). Although we demonstrated that a TPR domain other than the carboxylate clump region of FKBP8 was responsible for the specific interaction with NS5A (37
), the precise binding amino acid residue of the interaction was not determined. In the present study, FKBP8 exhibited a specific interaction with the immobilized NS5A in a dose-dependent manner with an equilibrium dissociation constant (Kd
) of 82 nM as determined by the SPR, but no interaction with FKBP52 was detected. Furthermore, mutational analysis suggested that Val or Ile at the amino acid residue 121 of NS5A was responsible for the specific interaction with FKBP8. The subgenomic HCV replicon RNA harboring the mutation of Val121
to Ala within NS5A leads to severe impairment of RNA replication, and reversion from Ala121
to Val was detected, suggesting that interaction of FKBP8 with NS5A through the Val121
is crucial for HCV replication. The crystal structure of NS5A domain 1 revealed that Val121
is located on one of the β-sheet structures in the 1B subdomain and the side chain of the residue is located within the hydrophobic core (46
); therefore, the Val121
may be involved in the maintenance of the β-sheet structure in the subdomain rather than the direct interaction with FKBP8. However, it remains feasible to speculate that unidentified host factors may be involved in the conformational change of region, including Val121
for direct interaction with FKBP8. Further studies, including a structural analysis of FKBP8, are needed to clarify the mechanisms by which HCV is replicated through the interaction of NS5A, FKBP8, and Hsp90.
The current combination therapy with pegylated IFN-α and ribavirin achieves a sustained virological response in half of the patients infected with a high viral load of HCV of genotype 1b (30
). However, it is difficult to achieve the complete removal of viruses by antiviral drugs targeted to the viral enzymes, including proteases and polymerases, from patients persistently infected with RNA viruses that exhibit a quasispecies nature, such as human immunodeficiency virus and HCV. Viral quasispecies are not a simple collection of diverse mutants but a group of interactive variants capable of adapting to new environments (48
). Cyclosporine treatment has been shown to be effective for patients infected with HCV of genotype 1b (20
) and suppresses HCV RNA replication in vitro (52
). In addition, cyclosporine has been shown to disrupt the interaction between NS5B and cyclophilin B, which is required for an efficient RNA-binding of NS5B (53
). Cyclophilins and FKBPs are classified as immunophilins capable of binding to the immunosuppressants cyclosporine and FK506, respectively (26
). The family members do not share a homologous domain other than drug-binding and enzymatically active domains, based on their amino acid sequences, substrate specificities, and inhibitor sensitivities. However, cyclosporine-resistant RNA replicon was shown to exhibit mutations not only in NS5B but also in NS5A (12
), suggesting that cyclosporine might affect the viral replication through the nucleotide-binding ability of NS5B, as well as the function of NS5A. Recently, geldanamycin, an inhibitor of Hsp90, was shown to drastically impair the replication of poliovirus without any emergence of escape mutants (14
). Therefore, the elucidation of host proteins, including immunophilins and chaperones, participating in the HCV replication complex may lead to the development of new therapeutics for chronic hepatitis C with a broad spectrum and a low possibility of emergence of revertant viruses. In particular, disruption of the specific interaction of Val121
of NS5A with the TPR domain of FKBP8 might be an ideal target for a novel therapeutic measure.
Egger et al. reported that NS4B alters the intracellular membrane to form a membranous web structure consisting of a membrane-associated multiprotein complex localized in the cytoplasmic compartments distinct from the mitochondria in vitro and in the liver of an HCV-infected chimpanzee, suggesting that the membranous web forms the viral replication complex (8
). An N-terminal amphipathic helix of NS4B plays an important role in the viral replication, as well as in the correct localization of other NS proteins including NS5A (9
). Furthermore, VAP-B was reported to interact with Nir2 protein through the FFAT (named for two phenylalanines [i.e., FF] in the acidic tract) motif and to remodel the ER structure to form a convoluted membrane structure resembling a membranous web (3
). In addition, VAP-A and B interact with not only NS5A but also NS5B (13
), suggesting that the complex of NS5A with FKBP8 might be recruited on the membranous web by NS4B and/or VAPs and participate in the HCV replication.
FKBP8 has been shown to be localized mainly on the mitochondria and to interact with Bcl-2 to sequester Bcl-2 on the mitochondria (7
). However, HCV RNA was suggested to be replicated in the membranous web structure in replicon cells (8
), and NS5A was reported to localize on the ER, Golgi apparatus (2
), and lipid droplets (43
). Figures and clearly indicate that the intracellular compartment including NS5A and FKBP8 is distinct from mitochondria. The HCV core protein was shown to upregulate genes related to fatty acid biosynthesis through the interaction with proteasome activator PA28γ/REGγ in the nucleus (34
) and to induce accumulation of cytoplasmic lipid droplets in the mouse liver (35
). Recently, it was shown that the HCV core protein of the genotype 2a JFH1 strain recruits the replication complex to the lipid droplet-associated membranes, and HCV particles were detected in close proximity to the lipid droplets, suggesting that lipid droplets induced by core protein participate in the assembly of HCV particles (31
). In addition, the lipid droplets including the core protein were surrounded by the nonstructural proteins was also detected in cells expressing the chimeric HCV genomes encoding core to a part of NS2 proteins of genotype 1b or 1a strain and the nonstructural proteins of JFH1 strain (31
). In the present study, FKBP8 was shown to be colocalized with NS5A in a highly electron-dense intracellular compartment indistinguishable from the membranous web. Although the total amount of FKBP8 was not changed by the treatment of the replicon cells by IFN-α (data not shown), the membranous web structure where FKBP8 and NS5A had accumulated was removed by the treatment (Fig. ). These results suggest that the replication of the subgenomic HCV RNA induces the formation of a membranous web structure in which NS5A and FKBP8 are colocalized but has no effect on the expression level of FKBP8. Furthermore, we could not detect any colocalization of FKBP8 and NS5A with the lipid droplets in the replicon cells harboring a full-length genome of the genotype 1b Con1 strain (data not shown). Although the relationships between the membranous web and lipid droplets remain unknown, these discrepancies might be attributable to the difference in HCV genotypes of the nonstructural proteins that consist of the major components of the replication complex determining the efficiency of HCV replication.
In conclusion, our data indicate that NS5A directly binds to FKBP8 through the Val121 and colocalizes in the convoluted membrane structure known as the membranous web. Future studies on the role of FKBP8 in the replication of HCV might contribute to the development of a new type of anti-HCV drugs with a low frequency of emergence of drug-resistant breakthrough viruses.