Interleukin-29 functions cooperatively with interferon to induce antiviral gene expression and inhibit hepatitis C virus replication

The interferon (IFN)-related cytokine interleukin (IL)-29 (also known as IFN-lambda1) inhibits virus replication by inducing a cellular antiviral response similar to that activated by IFN-alpha/beta. However, because it binds to a unique receptor, this cytokine may function cooperatively with IFN-alpha/beta or IFN-gamma during natural infections to inhibit virus replication, and might also be useful therapeutically in combination with other cytokines to treat chronic viral infections such as hepatitis C (HCV). We therefore investigated the ability of IL-29 and IFN-alpha or IFN-gamma to cooperatively inhibit virus replication and induce antiviral gene expression. Compared with the individual cytokines alone, the combination of IL-29 with IFN-alpha or IFN-gamma was more effective at blocking vesicular stomatitis virus and HCV replication, and this cooperative antiviral activity correlated with the magnitude of induced antiviral gene expression. Although the combined effects of IL-29 and IFN-alpha were primarily additive, the IL-29/IFN-gamma combination synergistically induced multiple genes and had the greatest antiviral activity. Two different mechanisms contributed to the enhanced gene expression induced by the cytokine combinations: increased activation of ISRE promoter elements and simultaneous activation of both ISRE and GAS elements within the same promoter. These findings provide new insight into the coregulation of a critical innate immune response by functionally distinct cytokine families.     

Lambda interferon inhibits hepatitis B and C virus replication

Lambda interferon (IFN-lambda) induces an intracellular IFN-alpha/beta-like antiviral response through a receptor complex distinct from the IFN-alpha/beta receptor. We therefore determined the ability of IFN-lambda to inhibit hepatitis B virus (HBV) and hepatitis C virus (HCV) replication. IFN-lambda inhibits HBV replication in a differentiated murine hepatocyte cell line with kinetics and efficiency similar to IFN-alpha/beta and does not require the expression of IFN-alpha/beta or IFN-gamma. Furthermore, IFN-lambda blocked the replication of a subgenomic and a full-length genomic HCV replicon in human hepatocyte Huh7 cells. These results suggest the possibility that IFN-lambda may be therapeutically useful in the treatment of chronic HBV or HCV infection.     

Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities

Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. The underlying mechanisms of chronic HCV infection and IFN-based therapies, however, have not been defined. Protein kinase R (PKR) was implicated in the control of HCV replication and mediation of IFN-induced antiviral response. In this report, we demonstrate that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs. Additionally, the subgenomic HCV RNA was found to replicate more efficiently in the PKR knockout (PKR(-/-)) MEF than in the wild-type (PKR(+/+)) MEF. The knockdown expression of PKR by specific small interfering RNAs significantly enhanced the level of HCV RNA replication, suggesting that PKR is involved in the control of HCV RNA replication. The level of ISG56 (p56) was induced by HCV RNA replication, indicating the activation of PKR-independent antiviral pathways. Furthermore, IFN-alpha/beta inhibited HCV RNA replication in PKR(-/-) MEFs as efficiently as in PKR(+/+) MEFs. These findings demonstrate that PKR-independent antiviral pathways play important roles in controlling HCV replication and mediating IFN-induced antiviral effect. Our findings also provide a foundation for the development of transgenic mouse models of HCV replication and set a stage to further define the roles of cellular genes in the establishment of chronic HCV infection and the mediation of intracellular innate antiviral response by using MEFs derived from diverse gene knockout animals.     

Immune-based novel therapies for chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a great public health problem, with an estimated 200 million chronically infected patients worldwide. No vaccines are currently available for HCV, and only a subset of HCV patients responds to interferon-alpha (IFN-alpha) and Ribavirin treatment. Substantial evidence has emerged recently to support the role of the host immune response in the outcome and pathogenesis of HCV infection. Our aims of this article are to present the immune-based novel therapeutic options for HCV infection and the evidence supporting their use in patients with chronic hepatitis C. There is a growing consensus that acute control of HCV infection is associated with a vigorous intrahepatic antiviral CD4+ and CD8+ T cell response. IFN-gamma was detectable in the livers of the chimpanzees that cleared or controlled the virus, raising the possibility that IFN-gamma might perform antiviral effector functions during HCV infection. Based on these observations, therapeutic induction of intrahepatic IFN-gamma by adoptive immunotherapy might be able to control chronic HCV infection. Immune-based novel therapies appear to hold great promise in treating chronic HCV infection.     

Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and gamma interferon (IFN-gamma) inhibit hepatitis B virus (HBV) replication by eliminating pregenomic RNA containing viral capsids from the hepatocyte. We have also shown that HBV-specific cytotoxic T lymphocytes that induce IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the liver can inhibit HBV gene expression by destabilizing preformed viral mRNA. In order to further study the antiviral activity of IFN-alpha/beta, IFN-gamma, and TNF-alpha at the molecular level, we sought to reproduce these observations in an in vitro system. Accordingly, hepatocytes were derived from the livers of HBV-transgenic mice that also expressed the constitutively active cytoplasmic domain of the human hepatocyte growth factor receptor (c-Met). Here, we show that the resultant well-differentiated, continuous hepatocyte cell lines (HBV-Met) replicate HBV and that viral replication in these cells is efficiently controlled by IFN-alpha/beta or IFN-gamma, which eliminate pregenomic RNA-containing capsids from the cells as they do in the liver. Furthermore, we demonstrate that IFN-gamma, but not IFN-alpha/beta, is capable of inhibiting HBV gene expression in this system, especially when it acts synergistically with TNF-alpha. These cells should facilitate the analysis of the intracellular signaling pathways and effector mechanisms responsible for these antiviral effects.     

Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo

Type III interferons (IFNs) (interleukin-28/29 or lambda interferon [IFN-lambda]) are cytokines with IFN-like activities. Here we show that several classes of viruses induce expression of IFN-lambda1 and -lambda2/3 in similar patterns. The IFN-lambdas were-unlike alpha/beta interferon (IFN-alpha/beta)-induced directly by stimulation with IFN-alpha or -lambda, thus identifying type III IFNs as IFN-stimulated genes. In vitro assays revealed that IFN-lambdas have appreciable antiviral activity against encephalomyocarditis virus (EMCV) but limited activity against herpes simplex virus type 2 (HSV-2), whereas IFN-alpha potently restricted both viruses. Using three murine models for generalized virus infections, we found that while recombinant IFN-alpha reduced the viral load after infection with EMCV, lymphocytic choriomeningitis virus (LCMV), and HSV-2, treatment with recombinant IFN-lambda in vivo did not affect viral load after infection with EMCV or LCMV but did reduce the hepatic viral titer of HSV-2. In a model for a localized HSV-2 infection, we further found that IFN-lambda completely blocked virus replication in the vaginal mucosa and totally prevented development of disease, in contrast to IFN-alpha, which had a more modest antiviral activity. Finally, pretreatment with IFN-lambda enhanced the levels of IFN-gamma in serum after HSV-2 infection. Thus, type III IFNs are expressed in response to most viruses and display potent antiviral activity in vivo against select viruses. The discrepancy between the observed antiviral activity in vitro and in vivo may suggest that IFN-lambda exerts a significant portion of its antiviral activity in vivo via stimulation of the immune system rather than through induction of the antiviral state.     

Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo

The innate immune response, and in particular the alpha/beta interferon (IFN-alpha/beta) system, plays a critical role in the control of viral infections. Interferons alpha and beta exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-alpha/beta. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-alpha/beta receptor (IFN-alpha/betaR)(-/-) mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.     

Adenovirus-mediated gene transfer of interferon alpha inhibits hepatitis C virus replication in hepatocytes

Recently we reported that on-site interferon (IFN)-alpha production in the liver using an adenovirus vector can achieve a substantial confinement of IFN-alpha in the target organ and can improve liver fibrosis in a rat liver cirrhosis model. However, the major therapeutic effect of IFN for hepatitis C virus (HCV)-associated liver diseases is its antiviral effect on HCV. As a prelude to the in vivo HCV infection experiment using a primate animal model, here we examined the antiviral effect of IFN-alpha gene transfer into HCV-positive hepatocytes in vitro. The non-neoplastic human hepatocyte cell line PH5CH8 was inoculated with HCV-positive serum. Successful in vitro HCV replication and thus the validity of this model was confirmed by a strong selection for HCV variants determined by sequence analysis of the hypervariable region and an increase of HCV RNA estimated by real time TaqMan RT-PCR. One day after the inoculation of HCV, PH5CH8 cells were infected with adenoviral vectors encoding human IFN-alpha cDNA. HCV completely disappeared 9 days after the adenoviral infection, which is linked to the increase of 2('),5(')-oligoadenylate synthetase activity, suggesting that IFN-alpha produced by gene transfer effectively inhibits HCV replication in hepatocytes. This study supports the development of IFN-alpha gene therapy for HCV-associated liver diseases.     

Alpha/beta interferons regulate murine gammaherpesvirus latent gene expression and reactivation from latency

Alpha/beta interferon (IFN-alpha/beta) protects the host from virus infection by inhibition of lytic virus replication in infected cells and modulation of the antiviral cell-mediated immune response. To determine whether IFN-alpha/beta also modulates the virus-host interaction during latent virus infection, we infected mice lacking the IFN-alpha/beta receptor (IFN-alpha/betaR(-/-)) and wild-type (wt; 129S2/SvPas) mice with murine gammaherpesvirus 68 (gammaHV68), a lymphotropic gamma-2-herpesvirus that establishes latent infection in B cells, macrophages, and dendritic cells. IFN-alpha/betaR(-/-) mice cleared low-dose intranasal gammaHV68 infection with wt kinetics and harbored essentially wt frequencies of latently infected cells in both peritoneum and spleen by 28 days postinfection. However, latent virus in peritoneal cells and splenocytes from IFN-alpha/betaR(-/-) mice reactivated ex vivo with >40-fold- and 5-fold-enhanced efficiency, respectively, compared to wt cells. Depletion of IFN-alpha/beta from wt mice during viral latency also significantly increased viral reactivation, demonstrating an antiviral function of IFN-alpha/beta during latency. Viral reactivation efficiency was temporally regulated in both wt and IFN-alpha/betaR(-/-) mice. The mechanism of IFN-alpha/betaR action was distinct from that of IFN-gammaR, since IFN-alpha/betaR(-/-) mice did not display persistent virus replication in vivo. Analysis of viral latent gene expression in vivo demonstrated specific upregulation of the latency-associated gene M2, which is required for efficient reactivation from latency, in IFN-alpha/betaR(-/-) splenocytes. These data demonstrate that an IFN-alpha/beta-induced pathway regulates gammaHV68 gene expression patterns during latent viral infection in vivo and that IFN-alpha/beta plays a critical role in inhibiting viral reactivation during latency.     

Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.     

Gene expression and antiviral activity of alpha/beta interferons and interleukin-29 in virus-infected human myeloid dendritic cells

Dendritic cells (DCs) respond to microbial infections by undergoing phenotypic maturation and by producing multiple cytokines. In the present study, we analyzed the ability of influenza A and Sendai viruses to induce DC maturation and activate tumor necrosis factor alpha (TNF-alpha), alpha/beta interferon (IFN-alpha/beta), and IFN-like interleukin-28A/B (IFN-lambda2/3) and IL-29 (IFN-lambda1) gene expression in human monocyte-derived myeloid DCs (mDC). The ability of influenza A virus to induce mDC maturation or enhance the expression of TNF-alpha, IFN-alpha/beta, interleukin-28 (IL-28), and IL-29 genes was limited, whereas Sendai virus efficiently induced mDC maturation and enhanced cytokine gene expression. Influenza A virus-induced expression of TNF-alpha, IFN-alpha, IFN-beta, IL-28, and IL-29 genes was, however, dramatically enhanced when cells were pretreated with IFN-alpha. IFN-alpha priming led to increased expression of Toll-like receptor 3 (TLR3), TLR7, TLR8, MyD88, TRIF, and IFN regulatory factor 7 (IRF7) genes and enhanced influenza-induced phosphorylation and DNA binding of IRF3. Influenza A virus also enhanced the binding of NF-kappaB to the respective NF-kappaB elements of the promoters of IFN-beta and IL-29 genes. In mDC IL-29 induced MxA protein expression and possessed antiviral activity against influenza A virus, although this activity was lower than that of IFN-alpha or IFN-beta. Our results show that in human mDCs viruses can readily induce the expression of IL-28 and IL-29 genes whose gene products are likely to contribute to the host antiviral response.     

Fas-associated death domain-containing protein-mediated antiviral innate immune signaling involves the regulation of Irf7

The induction of type I (alphabeta) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-beta and IFN-alpha4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-alpha subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-alphas and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-alpha production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd(-/-) murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD's antiviral activity involves regulation of IRF-7-dependent production of IFN-alpha subtypes and consequent induction of secondary antiviral genes.