Most HCV-infected individuals are unable to clear the virus and develop a chronic infection with inauspicious outcome. The mechanisms by which HCV resists the host antiviral defenses and induces liver injury remain poorly defined. Nevertheless, it is well established that HCV induces IFN, and the ability to circumvent the antiviral effects of this cytokine seems to be mainly responsible for the establishment of persistence. Accordingly, only 10% to 20% of patients with chronic infection respond to IFN-α therapy (51
). Thus, clarification of the mechanisms involved in impaired anti-HCV actions by IFN-α is an important goal for the definition of effective therapies.
In the present study we have demonstrated that HCV core gene expression, either alone or in the context of HCV replication, inhibits IRF-1, a transcription factor involved in IFN signaling. Furthermore, IRF-1 repression attenuates ISG responses mediated by the IRF-E/ISRE. Specifically, we showed that IRF-1 target genes, endowed with both antiviral and immunomodulatory functions, are repressed and IRF-1 suppression occurs at the transcriptional level through inhibition of both basal and cytokine-induced IRF-1 promoter activity.
IRF-1 is a pleiotropic transcription factor, critical for cell defense against viral infections but also crucial for the development of both the innate and adaptive responses. Indeed, the targets of IRF-1 during the antiviral response are genes directly involved not only in virus elimination but also in differentiation, proliferation activity, and apoptosis of cells, including those of the immune system (42
). Accordingly, dysregulation of IRF-1 expression can lead to defective antigen presentation, NK and T-cell activity disorders, and tumorigenesis.
The different levels of IRF-1 expression seem to be one of the factors that dictate how it functions. Expression of the IRF-1 gene is rapidly induced following virus infection, and IFNs, proinflammatory cytokines, and high IRF-1 expression levels are required for the induction of a set of ISGs necessary for an efficient antiviral response (24
). Here we have shown that HCV RNA replication affects IRF-1 expression, down-modulating both mRNA and protein at levels no more sufficient to induce the expression of ISGs direct targets of IRF-1.
The inhibition of IRF-1 expression and IRF-E/ISRE activity in cells expressing a subgenomic replicon of HCV was reported by Kanazawa and colleagues (30
). However, in the system analyzed, no direct correlation was found between the viral RNA replication and IRF-1 repression.
The development of in vitro culture systems for expression of the entire replicon of HCV greatly improved our understanding of the complex virus-host interactions and recapitulated most of the previous observations of the impact of single HCV proteins on cell physiology. In contrast to observations in HCV subgenome-expressing cells, we found that in cured Huh-7 cells from which the entire replicon has been eliminated, IRF-1 expression is restored (Fig. ), and we postulate that the decrease in IRF-1 expression could be due to active suppression by viral proteins. Indeed, using cells that transiently or conditionally express HCV structural proteins, we have demonstrated a specific repression of IRF-1 by the core protein.
Several in vitro studies have already defined the role of some viral structural and nonstructural proteins in inhibiting the expression and/or activity of key factors in IFN synthesis and in the IFN-induced signal transduction pathway (21
). Conversely, to date conflicting results have been reported on the role of HCV core protein. Whereas recent papers agree on the interference with the Jak/STAT pathway by inhibition of ISGF3 and STAT-1 activities, there is no agreement on whether this interference results in a modification of transcription of ISGs (3
). Variations in the experimental system used, structural or genotypic differences in the protein and, possibly, the amount of protein used may all be implicated in the reported discrepancies. Using conditional and transient expression of HCV structural proteins, we have shown that the core protein was mainly responsible for the inhibition of IRF-1 expression in cells bearing the entire HCV replicon. Conversely, and in agreement with previous reports (57
), other structural proteins, E1 and E2, did not impact IRF-1 expression (Fig. ).
We have also shown that the inhibition of IRF-1 expression occurred at the transcriptional level through both the STAT-1 and NF-κB consensus sequences on the IRF-1 promoter. In agreement with these data it has recently been reported that core protein inhibits both the activation and nuclear translocation of STAT-1 in IFN-treated cells (7
). In addition, in core-expressing cells NF-κB nuclear translocation and activity is also suppressed (29
). Data obtained in Vero cells, which are deficient in IFN production, seem also to exclude the possibility that the core-inhibited IRF-1 expression resulted from the blockade of the endogenous IFN in core-expressing cells. Given that no reports have, as yet, clarified the mechanism through which the core regulates gene transcription, our data support the conclusion that the core regulates gene transcription by means of indirect effects eventually leading to modulation of signal transduction and transcription factors.
Notably, we have also shown that the down-modulation of IRF-1 expression by the HCV replicon and by the core protein in turn affects host gene expression mediated by IRF-E/ISRE. Among the repressed genes, we found genes with antiviral functions, including 2-5A synthetase and PKR, but also immunomodulatory cytokines, such as IL-12 and IL-15, and genes that are important for antigen presentation, such as LMP2. These results suggest that inhibition of IRF-1 by the core in the context of HCV replication may trigger a cascade of events that affect a wide range of immune responses via direct or indirect mechanisms.
Numerous studies have shown that the HCV core protein is a major cause of most of the pathogenic features associated with HCV infection, and in this respect its role in the suppression of immunological functions has been established. In particular, impairment of the activation and functions of dendritic cells and of the proliferation of T cells has been reported (25
). In accordance with our observations, a selective suppression of IL-12 in stimulated human macrophages mediated by the suppression of AP1 has also recently been described (15
). Our data also indicate that the HCV core protein affects IL-12 p40 gene promoter activity in both hepatoma and T cells. In this respect it has been demonstrated that induction of the gene encoding both the p40 and p35 subunits of IL-12 is totally dependent on IRF-1 (41
). Therefore, our results imply that in addition to the reported mechanism of IL-12 repression in macrophages mediated by AP-1 (15
), the inhibition of IRF-1 is probably most responsible for IL-12 suppression by the HCV core protein.
IL-12 is known to play a pivotal role in the generation of Th1 immune responses and provides a crucial link between innate and adaptive immunity (74
). IRF-1 has been defined as a “super” Th1-cell transcription factor (42
) in that IRF-1−/−
mice display completely defective Th1 responses due to a lack of IL-12 production by APC, accompanied by exclusive Th2 differentiation. In this respect, it is interesting that the study of IRF-1 promoter polymorphisms in relation to the response to IFN in chronic patients indicated that a lower viral load corresponded to higher IRF-1 promoter activity and to a significantly higher proportion of Th1 CD4+
cells after IFN administration (63
). Interestingly, the establishment of a Th1 environment in HCV-infected individuals is thought to be important in determining the outcome of the infection. As indicated by in vivo studies, patients who demonstrate Th2 dominance tend to develop chronic infection, whereas those with a Th1 phenotype can clear the virus (2
). The immunosuppressive action of core protein may thus be pivotal in viral escape by down-modulating Th1 responses in favor of Th2 responses, a mechanism in which core-mediated inhibition of IRF-1 may be instrumental.
Of the ISGs analyzed, IL-15, a unique target of IRF-1, was also substantially reduced in both hepatic and T cells expressing either the entire HCV replicon or the structural proteins, and to our knowledge this is the first study reporting a repression of IL-15 in cells bearing the entire HCV replicon. IL-15 is involved in the activation and homeostatic maintenance of cells of both the innate and adaptive immune systems, playing a key role in CD8+
T-cell homeostasis by promoting survival or proliferation of naive and memory phenotype CD8 T cells and in NK cell development, maturation, and survival (14
). Notably, it has been reported that impaired IL-15 production is one of the mechanisms of the aberrant response of DC to IFN in HCV-infected patients (28
). In addition, a significant reduction in serum IL-15 levels in HCV patients has also been recently reported (46
We have also shown that the HCV core, through inhibition of IRF-1, specifically inhibits, at the transcriptional level, the LMP2 gene promoter activity (Fig. ). LMP2 is a subunit of immunoproteasomes, which are very efficient for the generation of specific CTL epitopes. It has, in fact, been shown that substitution of standard β-subunits of the proteasome with LMP2, LMP7, and MECL1 subunits improves the production of peptide antigens with the correct C termini for binding to MHC class I (53
). Since both basal and IFN-γ-induced LMP2 expression is absolutely dependent on IRF-1, it is not surprising that inhibition of IRF-1 by the core protein also results in suppression of LMP2 expression. Considering that a reduction in the population and specific activity of CTL (2
) have been observed in chronically infected individuals, we speculate that the HCV core protein down-modulation of LMP2 may be partially responsible for these in vivo observations. An analysis of the proteasome composition in HCV patients would, therefore, be very useful.
Data reported in the present study suggest that HCV regulation of IRF-1 may also impact other IRF-regulated pathways influencing host gene expression on a more global scale. In this respect, given that IRF-1 together with NF-κB is induced and is also an effector of inflammatory cytokines, it is interesting that the HCV core protein inhibits IRF-1 promoter activity even after treatment with inflammatory cytokines (Fig. ). This raises the possibility that both locally, in hepatoma cells, and systemically, by affecting PBMC, the core protein can depress the inflammatory response induced by virus infection also through repression of IRF-1. In agreement with this hypothesis, a recent report (27
) indicated that cyclooxygenase 2, an enzyme that contributes to homeostasis and to inflammatory pathways (56
), is substantially repressed in core-expressing cells. Notably, cyclooxygenase 2 is another gene tightly regulated by IRF-1 (5
Finally, it has been reported that other cellular and viral genes altered by the core include p21waf1
, and human immunodeficiency virus type 1 LTR
). As others and we ourselves have reported (65
), these genes are all specific targets of IRF-1.
Taking all these results into account and considering that they well mirror clinical observations in chronically infected patients, we conclude that IRF-1 repression by the HCV core protein can be considered a unifying mechanism that recapitulates most of the data so far reported on the dysregulation of cellular processes induced by this protein and may at least partially account for its role in evading the host response at several levels: antiviral, inflammatory, and immune. Restoration of the correct expression of IRF-1 in HCV-infected cells could, therefore, represent a new avenue for therapeutic interventions.