The majority of HCV-infected individuals become chronic carriers; however, the mechanism of progression to chronicity remains unclear. Among HCV proteins, NS3 has been shown to be immunodominant, and T cells that are reactive to NS3 have been suggested to play a crucial role in viral clearance, while HCV core protein is immunosuppressive (8
). Treatment of immature DCs with core or NS3 protein inhibited DC differentiation, and DCs transduced to express core or E1 protein exhibited poor allogeneic T-cell responses (43
). The immunosuppressive potential of HCV proteins has been implicated as a mechanism of the functional subversion of T cells, natural killer (NK) cells, and DCs. The association of HCV core protein with the globular domain of the C1q receptor on T cells down-regulates T-cell proliferation and IL-2 production (25
). Additionally, the HCV E2 protein displays a high affinity for the tetraspanin cell surface molecule human CD81, which is one of the candidates for an HCV entry receptor (40
), and E2 cross-linking with cell surface human CD81 impairs the activation of NK cells (7
In the present study, we established macrophage cell lines stably expressing HCV proteins and examined the effects of viral proteins on TLR function. The expression of the NS5A protein specifically inhibits TLR-MyD88-induced signaling by associating with the death domain of MyD88 through the ISDR spanning amino acid residues 240 to 280 in macrophage cells. HCV NS5A is a phosphoprotein that appears to possess multiple and diverse functions in viral replication, IFN resistance, and pathogenesis (34
). Mutation in the ISDR has been suggested to correlate with the responsiveness of patients chronically infected with HCV genotype 1b to IFN treatment (10
). Furthermore, NS5A has been shown to rescue virus replication in IFN-treated cell cultures (41
) and to inhibit the antiviral activity of IFN by binding to PKR through the ISDR and its adjacent region (amino acids 237 to 302) (13
). However, controversial observations that the ISDR sequence variation does not account for differences in IFN sensitivity in patients (9
) and also in an HCV subgenomic RNA replicon system (15
) have been made. Moreover, the expression of NS5A or the entire HCV polyprotein has been reported to counteract the antiviral effect of IFN in a PKR- and ISDR-independent manner (12
). Therefore, the possibility remains that a molecule other than PKR may be involved in the NS5A-mediated inhibition of IFN (50
). Restoration of the phosphorylation of STAT1 in cells expressing a deletion mutant lacking ISDR in response to IFN-α and that of PKR phosphorylation upon infection with VSV in cells expressing NS5A mutants lacking amino acid residues 240 to 280 (ISDR) or 280 to 300 may support the hypothesis that the ISDR and the adjacent region are involved in IFN sensitivity. Thus, the ISDR may participate not only in conferring IFN resistance but also in disrupting TLR-MyD88 signaling pathways in macrophage cells.
Several viral proteins have been shown to counteract TLRs and their downstream signaling cascade. The vaccinia virus A46R protein contains a Toll/IL-1 receptor domain that interacts with multiple Toll/IL-1 receptor-containing adaptor molecules, thereby inhibiting the activation of NF-κB and IRF3 (49
). Measles virus and respiratory syncytial virus have been shown to inhibit the TLR7- and TLR9-dependent IFN-inducing pathways stimulated by R848 and CpG oligodeoxynucleotides in primary human pDCs (45
). HCV NS3/4A has been shown to influence the functions of adaptor molecules mediating TLR-dependent and -independent signaling pathways, resulting in an impairment of the induction of IFN-β as well as the subsequent IFN-inducible genes (11
). Recently, RIG-I and MDA5 have been identified as being cytoplasmic dsRNA detectors responding to viral RNAs and poly(I:C) in a TLR-independent manner and recruit IPS-1 as an adaptor molecule for signal transduction (24
). The uncapped 5′-triphosphate RNA generated by viral polymerases was shown to be selectively recognized by RIG-I (18
). In this study, we could demonstrate that the invasion of VSV and poly(I:C) into RAW cell lines is preferentially recognized by RIG-1-IPS-1- and TLR3-TRIF-dependent signaling pathways, respectively, and that the expression of HCV NS3/4A protease selectively inhibits cytokine production upon infection with VSV through the cleavage of IPS-1. Therefore, it is feasible that the expression of NS5A and NS3/4A proteins in macrophage cells may disrupt TLR-dependent and -independent signaling pathways, respectively. However, the mechanism for the inhibition of the TLR signaling pathway in the macrophage cells by the expression of NS3 or NS4B remains unclear.
Although there have been reports suggesting a lack of DC dysfunction in both chimpanzees and humans chronically infected with HCV (26
), direct infection of DCs with HCV may be a plausible mechanism for the dysfunction of DCs in patients with chronic HCV infection (4
). Indeed, the HCV genome has been detected in DCs by PCR (4
), and HCV was detected in a monocyte/macrophage subpopulation of peripheral blood mononuclear cells from patients with chronic HCV infection (5
). Further experiments are needed to exclude the possibility of contamination of viral RNA in blood samples. Pseudotype VSV-bearing chimeric HCV E1 and E2 proteins have been shown to infect immature myeloid DCs isolated from healthy donors through interactions with lectins in a Ca-independent manner (20
). Recently, the in vitro replication of the HCV JFH1 clone of genotype 2a isolated from an HCV-infected patient who developed fulminant hepatitis was reported (31
). However, in vitro replication was limited in the combination of HCV clones derived from strain JFH1 and certain human hepatoma cell lines, and a robust cell culture of genotypes 1a and 1b, the most prevalent viruses in the world and resistant to IFN therapy, has not yet been successful except for a cell culture system for strain H77-S (genotype 1a) in which infectivity was significantly lower than that of the JFH1 clone (56
). The establishment of a robust and reliable in vitro replication system for various HCV isolates is essential to determine the role of HCV infection in the modulation of TLR function in immunocompetent cells.
In conclusion, we have shown that the expression of the HCV nonstructural protein NS3, NS3/4A, NS4B, or NS5A impairs the activation of TLR signaling pathways in immunocompetent cells. Furthermore, the NS5A protein was shown to inhibit the TLR-MyD88 signaling pathway by a direct interaction with the death domain of MyD88 through the ISDR. These findings suggest new aspects of virus-cell interactions that may be explored to develop a greater understand of the mechanisms of escape of HCV from the host immune surveillance system and the establishment of persistent infection. However, it remains to be proven whether the results obtained using murine macrophage cell lines are applicable to immunocompetent cells in patients with HCV infection.