Antiviral therapy for HCV infection is still rather primitive, with only two Food and Drug Administration-approved drugs available. While newer-generation therapies for HCV are clearly more effective than conventional IFN monotherapy, over 60% of patients with HCV genotype 1 infection are not cured of their infection. In order to improve the success of antiviral intervention in HCV, it is important to understand the molecular mechanisms of antiviral resistance. In recent years, insight into the molecular mechanisms involved in the intrinsic resistance of HCV to IFN therapy has been gained. The emerging trend in the literature is that HCV proteins, when overexpressed in vitro, disrupt normal biological processes by interaction with cellular proteins. Two examples of this is the interaction of HCV NS5A and E2 proteins with the IFN-induced enzyme PKR (19
). However, since the roles of mutations in the NS5A protein and in the clinical response to IFN therapy and the effects of NS5A on the IFN system in vitro are controversial, it has been suggested that NS5A may use other mechanisms to perturb the IFN-induced antiviral response (59
The present study provides detailed mechanistic evaluation of an HCV protein in the context of the broad IFN response pathway. The present report found no evidence for a role of NS5A in inhibiting signal transduction via the STAT/JAK pathway, at least in terms of tyrosine phosphorylation of STAT-1 and induction of ISGF-3, the principal factor induced by IFN. This finding was supported by the observations that NS5A did not affect the induction of expression of two IFN-induced genes, those for PKR and MHC antigen. It is possible that levels of serine phosphorylation on STAT proteins are affected by NS5A expression (78
). Evidence was provided that NS5A can induce the expression of IL-8 mRNA and protein. IL-8 expression was associated with NS5A-induced inhibition of the IFN-α antiviral response in vitro, and the addition of recombinant human IL-8 also partially rescued EMCV replication during IFN challenge. Using a luciferase reporter gene under the control of the IL-8 promoter, it was also shown that NS5A expression could transactivate the IL-8 promoter.
It is currently unknown if NS5A-mediated transcriptional activation of the IL-8 promoter occurs directly or indirectly. NS5A may in theory bind to the IL-8 promoter if it can enter the nucleus. Evidence discounting a direct effect of NS5A are derived from observations that full-length NS5A proteins have a cytoplasmic perinuclear distribution when expressed in human cells (29
). It is possible, however, that a small amount of the NS5A protein, beyond the level of detection in these systems, may migrate to the nucleus. In this regard, it has recently been shown that processing of NS5A occurs naturally in transfected cells in vitro and that N-terminally deleted forms of the protein migrate to the nucleus (68
). The C terminus of NS5A also contains a nuclear localization signal that does not by itself direct NS5A to the nucleus but is nonetheless functional in directing nuclear translocation when it is placed at the amino terminus of a reporter gene (29
). In agreement with previous reports, we found that deletion of the amino terminus from NS5A resulted in increased transactivation of the IL-8 promoter, which was associated with increased nuclear staining of the protein. Deletion of C-terminal amino acids from NS5A created a protein that failed to transactivate the IL-8 promoter, and the protein appeared to be retained in the cytoplasm. These results suggest that the NS5A protein may be able to enter the nucleus under certain physiological instances.
Promoter mutagenesis experiments suggested that the minimal region for NS5A transactivation involved the 133 bp of the IL-8 promoter. Previous reports indicate that the transcription factors NF-κB, AP-1, and NF–IL-6 are involved in TNF-α-induced stimulation of the IL-8 promoter and that the involvement of each transcription factor can be cell line dependent (49
). In this report, we found that TNF-α induction of the IL-8 promoter in HeLa cells required NF-κB and AP-1 but that NF–IL-6 appeared inhibitory to this process. NS5A expression could not overcome deletion of the NF-κB and AP-1 binding sites to activate luciferase activity. Thus, it is possible that NS5A induces IL-8 transcription by interacting with transcription factors. Indeed, NS5A has been shown to interact with a novel transcription factor (23
). We are investigating further the domains of NS5A responsible for IL-8 transactivation and the subcellular localizations of the proteins and characterizing the transcription factors required for this process.
The results presented in this and other reports (17
) suggest that NS5A is a pleiotropic molecule, possessing multiple activities in vivo through its interaction with various cellular proteins and signaling pathways. Specifically, it appears that NS5A employs multiple mechanisms to achieve antiviral resistance, implying that inhibition of PKR by NS5A is but one way by which NS5A subverts the IFN system. This may be a plausible hypothesis since NS5A-ISDR mutations are not universally associated with IFN sensitivity (7
). More recent data suggest that a region in the C terminus of NS5A, termed the variable 3 (V3) region, may be associated with IFN resistance, at least in France and North America (11
), and with a distinct region within the proline-rich domain of NS5A (52
). A recent study also demonstrated that expression of the entire HCV polyprotein inhibits the activity of IFN against EMCV independently of PKR (14
). Moreover, HCV replicons selected for increased replicative ability in vitro harbor mutations in NS5A, including a deletion of the entire ISDR (3
). It should be emphasized that NS5A gene-encoded mechanisms for inhibition of the IFN system need not be mutually exclusive. It is possible that the different activities of NS5A are related to the location of the protein in the cell. In this scenario, cytoplasmic NS5A may inhibit PKR while nuclear NS5A may interact with transcription factors and influence gene expression.
Why NS5A would upregulate a chemokine that induces an inflammatory response is currently not known. One might speculate that NS5A-induced increases in IL-8 affects the activity of the intrahepatic immune response to HCV, through T-cell chemotaxis to the HCV-infected liver. Since T-cell infiltration is a hallmark histological finding of chronic hepatitis C, NS5A induction of IL-8 may ultimately be involved in HCV persistence and pathogenesis. Since IL-8 is elevated in alcoholic hepatitis (44
), it is tempting to speculate that NS5A induction of IL-8 may exacerbate the deleterious effects of ethanol on the liver, contributing to the increase in liver disease activity and poor antiviral responses in HCV-infected patients who abuse alcohol (69
). Moreover, since lymphocytes have been shown to harbor replicating HCV (37
), NS5A induction of IL-8, followed by lymphocyte recruitment to the infected liver, may also facilitate the spread of HCV to extrahepatic reservoirs. It is also possible that NS5A-mediated induction of IL-8 may contribute to HCV antiviral resistance, through the interaction between the chemokine- and IFN-signaling pathways. Indeed, there are several reports of IFN-induced downregulation of IL-8 (2
). Thus, NS5A may subvert the IFN signaling pathway to promote the expression of cellular genes that may counteract the IFN-induced antiviral response. These concepts will remain speculative until they can be formally addressed in a small animal model or in in vitro expression and replication systems.
Currently, the mechanism of IL-8-mediated inhibition of the IFN system is not known. Khabar and colleagues (33
) demonstrated that the anti-IFN effect of IL-8 was detectable as late as 20 h after the addition of IFN. This suggests that IL-8 inhibits the IFN-induced antiviral response at a posttranscriptional level and is consistent with this report showing that NS5A does not affect IFN signal transduction. Preliminary data suggest that IL-8 inhibited 2′-5′-oligoadenylate synthetase (OAS), a major pathway for IFN action against RNA viruses (34
). We are currently exploring the hypothesis that NS5A induction of IL-8 perturbs the OAS and other IFN-induced effector molecule pathways. It is also possible that the anti-IFN effect of IL-8 involves the high-effinity IL-8 receptor CXCR1. Indeed, HeLa cells which are susceptible to IL-8 proviral action express CXCR1 but not CXCR2 (K. S. A. Khabar, unpublished data; 34
Viral modulation of chemokine expression represents the continuous battle between viral invaders and antiviral, inflammatory, and immune responses. Perhaps the best examples are the recent discoveries that human immunodeficiency virus (HIV) entry into macrophages and T lymphocytes requires interaction with distinct chemokine receptors and that soluble chemokines can inhibit the entry of HIV (20
). In HIV infection, IL-8 is frequently elevated and the HIV Tat and Vpr proteins can induce IL-8 expression (54
). Other examples include human cytomegalovirus, which encodes a chemokine receptor that may facilitate viral replication by regulating cell cycle progression or inhibiting apoptosis (51
). Recent studies have demonstrated that Sendai virus-infected cells induce the CC chemokine RANTES and that this effect is mediated by interferon regulatory factor 3 and NF-κB (21
). Thus, it will be important to determine the mechanisms involved in the induction of IL-8 by the HCV NS5A protein and how IL-8 inhibits the antiviral actions of IFN. If this interaction between HCV and the chemokine system can be proven to occur in vivo, then its selective disruption may increase therapeutic response rates and reduce the pathogenicity associated with chronic hepatitis C.