Antiviral effect of interferon lambda against West Nile virus.
ABSTRACT: Type III interferons (IFN), IFN-lambda or IL-28/29, are new members of the IFN super-family. Except for using distinct receptors, type I and type III IFNs share the same major post receptor signaling components to activate the transcription of a similar set of IFN-stimulated genes (ISGs). To examine the antiviral effects of the new type IFNs against West Nile virus (WNV), we compared the antiviral effects of IFN-alpha and IFN-lambda on WNV virus-like particle (VLP) infection and replicon replication in Huh7.5 and Hela cells. The results revealed that (i) both types of IFNs could efficiently prevent the WNV infection, but IFN-alpha demonstrated a stronger antiviral efficacy; (ii) WNV genome replication in VLP-infected cells and replicon-containing cell lines could only be inhibited by IFN-alpha, but not IFN-lambda; (iii) in agreement with the observed antiviral effects, only IFN-lambda-induced activation of JAK-STAT signaling pathway and induction of ISG expression were completely inhibited in WNV replicon-containing cell lines, but IFN-alpha signal transduction was either unaffected or only partially inhibited in Huh7.5 or Hela cells by the virus. Hence, the differential inhibition of WNV on IFN-alpha and IFN-lambda signal transduction implies that the receptors of the two types of IFNs, but not the common post receptor signaling components, could be selectively targeted either directly by WNV nonstructural proteins or indirectly by the cellular responses induced by the virus infection to inhibit the signal transduction of the cytokines.
Project description: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.
Project description:Newcastle disease virus (NDV) strongly induces both type I and III antiviral interferons (IFNs-α/-β and IFN-λ, respectively) in tumor cells while it induces mainly type III IFN in normal cells. Impairment of antiviral type I IFN signaling in tumor cells is thought to be the reason for effective oncolysis. However, there is lack of clarity why lentogenic strain NDV can also induce oncolysis. NDV infection caused apoptosis in normal and tumor cells as demonstrated with the caspase-3 enzyme activation and annexin-V detection. The apoptosis response was inhibited by B18R protein (a type I IFN inhibitor) in tumor cells i.e. A549 and U87MG, and not in normal cells i.e. NB1RGB and HEK293. Similarly, UV-inactivated medium from NDV infection was shown to induce apoptosis in corresponding cells and the response was inhibited in A549 and U87MG cells with the addition of B18R protein. Treatment with combination of IFNs-α/-β/-λ or IFNs-α/-β or IFN-λ in NB1RGB, HEK293, A549 and U87MG showed that caspase activity in IFNs-α/-β/-λ group was the highest, followed with IFN-α/-β group and IFN-λ group. This suggests that tumor-selectivity of NDV is mainly because of the cumulative effect of type I and III in tumor cells that lead to higher apoptotic effect.
Project description:Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-alpha for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-alpha is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.
Project description:The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-alpha), IFN-gamma, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-alpha, IFN-gamma, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-alpha and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.
Project description:Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-alpha) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-alpha toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-lambda) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-lambda treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-alpha and IFN-lambda in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-lambda (BNL.IFN-lambda cells) or IFN-alpha (BNL.IFN-alpha cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-lambda and BNL.IFN-alpha cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-lambda. There was also a marked NK cell infiltration in IFN-lambda producing tumors. In addition, IFN-lambda and, to a lesser extent, IFN-alpha enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-gamma, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD11c+ and mPDCA+ dendritic cells responded directly to IFN-lambda. The antitumor activities of IFN-lambda against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-lambda to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer.
Project description:The clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-?1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alpha interferon (referred to herein as alfa). In the study described in this report, an assessment of the in vitro antiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-?1 (rIFN-?1) exerted the most robust effect, while rIFN-?3 exhibited greater activity than rIFN-?2. More importantly, cross-resistance to rIFN-?1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-?, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-?1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-?1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.
Project description:The type III interferon (IFN) family elicits an antiviral response that is nearly identical to that evoked by IFN-alpha/beta. However, these cytokines (known as IFN-lambda1, 2, and 3) signal through a distinct receptor, and thus may be resistant to the evasion strategies used by some viruses to avoid the IFN-alpha/beta response. Orthopoxviruses are highly resistant to IFN-alpha/beta because they encode well-characterized immunomodulatory proteins that inhibit IFN activity. These include a secreted receptor (B18R) that neutralizes IFN-alpha/beta, and a cytoplasmic protein (E3L) that blocks IFN-alpha/beta effector functions in infected cells. We therefore determined the ability of these immunomodulators to abrogate the IFN-lambda-induced antiviral response. We found that (i) vaccinia virus (VACV) replication is resistant to IFN-lambda antiviral activity; (ii) neither VACV B18R nor the variola virus homolog B20R neutralizes IFN-lambda; (iii) VACV E3L inhibits the IFN-lambda-mediated antiviral response through a PKR-dependent pathway; (iv) VACV infection inhibits IFN-lambdaR-mediated signal transduction and gene expression. These results demonstrate differential sensitivity of IFN-lambda to multiple distinct evasion mechanisms employed by a single virus.
Project description:Although type III interferons (IFN), also known as IFN-λ or IL28/IL-29, restrict infection by several viruses, their mechanism of inhibitory action has remained uncertain. We used recombinant IFN-λ and mice lacking the IFN-λ receptor (IFNLR1) to evaluate the effect of IFN-λ on infection with West Nile virus (WNV), an encephalitic flavivirus. Cell culture studies in keratinocytes and dendritic cells showed no direct antiviral effect of exogenous IFN-λ even though ISGs were induced. Correspondingly, we observed no differences in WNV burden between wild-type and Ifnlr1-/- mice in the draining lymph node, spleen, and blood. However, we detected earlier dissemination and increased WNV infection in the brain and spinal cord of Ifnlr1-/- mice, yet this was not associated with a direct antiviral effect on infection of neurons. Instead, an increase in blood-brain barrier (BBB) permeability was observed in Ifnlr1-/- mice. Accordingly, treatment of mice with pegylated IFN-λ2 resulted in decreased BBB permeability, reduced WNV infection in the brain without impacting viremia, and improved survival against lethal virus challenge. An in vitro model of the BBB showed that IFN-λ signaling in brain microvascular endothelial cells increased transendothelial electrical resistance, decreased virus movement across the barrier, and modulated tight junction protein localization in a protein synthesis- and STAT1-independent manner. Our data establish a novel indirect antiviral function of IFN-λ in which non-canonical signaling through IFNLR1 tightens the BBB and restricts viral neuroinvasion and pathogenesis. This finding suggests new clinical applications for IFN-λ in treating viral or autoimmune diseases. Overall design: Transcriptome profiling of bone-marrow derived Dendritic cells(BMDCs), treated with either Serum Free Media(Mock), interferon beta(IFNb), or interferon lambda(IFNL) for 6 hours.
Project description:Interferons (IFNs) play a major role in orchestrating the innate immune response toward viruses in vertebrates, and their defining characteristic is their ability to induce an antiviral state in responsive cells. Interferons have been reported in a multitude of species, from bony fish to mammals. However, our current knowledge about the molecular function of fish IFNs as well as their evolutionary relationship to tetrapod IFNs is limited. Here we establish the three-dimensional (3D) structure of zebrafish IFN?1 and IFN?2 by crystallography. These high-resolution structures offer the first structural insight into fish cytokines. Tetrapods possess two types of IFNs that play an immediate antiviral role: type I IFNs (e.g., alpha interferon [IFN-?] and beta interferon [IFN-?]) and type III IFNs (lambda interferon [IFN-?]), and each type is characterized by its specific receptor usage. Similarly, two groups of antiviral IFNs with distinct receptors exist in fish, including zebrafish. IFN?1 and IFN?2 represent group I and group II IFNs, respectively. Nevertheless, both structures reported here reveal a characteristic type I IFN architecture with a straight F helix, as opposed to the remaining class II cytokines, including IFN-?, where helix F contains a characteristic bend. Phylogenetic trees derived from structure-guided multiple alignments confirmed that both groups of fish IFNs are evolutionarily closer to type I than to type III tetrapod IFNs. Thus, these fish IFNs belong to the type I IFN family. Our results also imply that a dual antiviral IFN system has arisen twice during vertebrate evolution.
Project description:Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-alpha/beta and -lambda. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-alpha/beta and -lambda were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-beta and -lambda were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-alpha/beta and -lambda production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.