Respiratory syncytial virus strain A2 is resistant to the antiviral effects of type I interferons and human MxA.

Respiratory syncytial virus (RSV) belongs to Paramyxoviridae family of enveloped negative-strand RNA viruses and causes severe bronchiolitis and pneumonia in children younger than 2 years of age. As members of Paramyxoviridae family, RSV and parainfluenza type 3 (PIV3) have similar modes of infection and replication. A variety of negative-strand RNA virus infections, including that of PIV3, are inhibited by human MxA protein, a type I interferon (IFN)-inducible GTPase. We tested whether the MxA protein, induced either by type I human IFNs or by stable transfection of human MxA gene in human (U-87) or simian (Vero) cells, confers resistance to these cells against infection by RSV strain A2. RSV infection was resistant to antiviral effects induced by 0-10,000 U/ml type I IFNs (IFN-alpha or -beta) in both human lung epithelial, A549, and fibroblast, MRC-5 cells. RSV virus yield was reduced only by 10- to 20-fold, and viral protein synthesis was not significantly affected under conditions of IFN treatment where PIV3 yield was reduced by 1000- to 10,000-fold. Human or simian cell lines constitutively expressing MxA were protected against infection by PIV3 but not by RSV. Our results indicate that RSV A2 is resistant to the antiviral effects of MxA, even though RSV and PIV3 have similar replication strategies. In IFN-treated coinfected cultures, IFN-resistant RSV A2 did not prevent the IFN-mediated inhibition of PIV3 multiplication. Hence the resistance of RSV A2 to type I IFNs does not appear to be due to soluble factors released into the medium or a disruption in the cellular antiviral machinery brought about by RSV A2 infection.     

MxA-independent inhibition of Hantaan virus replication induced by type I and type II interferon in vitro

Interferons (IFN) induce an antiviral state against Hantaan virus (HTNV) but the mechanisms responsible for inhibition are unclear. The IFN-inducible MxA is discussed to be important for control of infections with hantaviruses. To characterize the role of endogenous MxA, the inhibition of HTNV induced by type I and type II IFNs was compared in Vero and A549 cells. IFNalpha and IFNgamma reduced production of infectious virions, viral RNA, and nucleocapsid protein with the same efficiency, although expression of MxA protein was detectable only in IFNalpha-treated A549 cells. Furthermore, knock down of MxA expression did not impair IFNalpha-induced inhibition. Thus, inhibition of HTNV induced by type I and type II IFNs did not dependent on expression of endogenous MxA. Taken together, these data suggest that MxA endogenously expressed in response to type I or type II IFNs does not play a pivotal role for the antiviral state against HTNV and that there is more than one mechanism by which cellular defences block hantavirus replication.     

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.     

Systemic responses during local viral infections: type I IFNs sound the alarm

Type I IFNs are well known for their role in controlling virus replication and spread. Type I IFNs produced by the infected tissue also signal beyond the boundaries of the infection to regulate different elements of the anti-viral immune response. Recent reports show that type I IFNs directly condition naive monocytes residing in the distal bone marrow (BM) and induce the expression of effector molecules in memory T cells, before their recruitment to the infected site. In addition, hematopoietic stem cells (HSCs) were shown to enter the cell cycle in response to systemically distributed type I IFNs. These discoveries expand our understanding of the pleiotropic effects of type I IFNs during infection and highlight the critical role of systemic signals in the development of an effective response to a localized viral infection.     

Hepatitis C virus NS3/4A protease blocks IL-28 production

Type I interferons (IFNs), including IFN-α, -β, and -ω, play a critical role in innate immune responses against viral infection. IFN-λ, including IL-29, IL-28A, and IL-28B, recently identified as a new subfamily of IFN named type III IFN, has also been demonstrated to suppress virus replication in vitro and in vivo. However, the molecular mechanisms that regulate the induction of type III IFNs during viral infection remain elusive. Here, we demonstrate that IL-28 (IFN-λ 2/3) IFN production, similar to type I IFN, represents a primary and direct host response to HCV genomic RNA transfection. IL-28 (IFN-λ2/3) induction by HCV genomic RNA was dependent upon the activation of NF-κB and IRF3. We identified a minimal IL-28 promoter region consisting of putative NF-κB and IRF3-binding sites. Furthermore, we showed that HCV infection can inhibit HCV genomic RNA-induced IL-28 expression, and that the viral NS3/4A protease activity was responsible for this inhibitory effect. Our results present important evidence for the control of type III IFN response by HCV, and shed more light on the molecular mechanisms underlying the persistence of HCV infection.     

Herpes simplex virus-1 infection causes the secretion of a type I interferon-antagonizing protein and inhibits signaling at or before Jak-1 activation

Host cells respond to viral infection by the production of type I interferons (IFNs), which induce the expression of antiviral genes. Herpes simplex virus I (HSV-1) encodes many mechanisms that inhibit the type I IFN response, including the ICP27-dependent inhibition of type I IFN signaling. Here we show inhibition of Stat-1 nuclear accumulation in cells that express ICP27. ICP27 expression also induces the secretion of a small, heat-stable type I IFN antagonizing protein that inhibits Stat-1 nuclear accumulation. We show that the inhibition of IFN-induced Stat-1 phosphorylation occurs at or upstream of Jak-1 phosphorylation. Finally, we show that ISG15 expression is induced after IFNα treatment in mock-infected cells, but not cells infected with WT HSV-1 or ICP27⁻ HSV-1. These data suggest that HSV-1 has evolved multiple mechanisms to inhibit IFN signaling not only in infected cells, but also in neighboring cells, thereby allowing for increased viral replication and spread.     

Recombinant chicken interferon-alpha inhibits the replication of exogenous avian leukosis virus (ALV) in DF-1 cells

Chickeninterferon alpha (ChIFNα) belongs to type I IFNs that are important antiviral cytokines. We investigated whether ChIFNα plays a role in avian leukosis virus (ALV) infections of chickens. Firstly, we explored the immune response to ALV in vivo by measuring cytokine expression profiles in the spleens and bursas of chickens during the late stages of ALV-J infection. The results indicated that ALV-J infection could induce a mixed Th1/Th2 cytokine response by elevating levels of both interleukin-2 (IL-2) and IL-10. In contrast, tumor necrosis factor alpha (TNF-α) levels decreased in the spleen while interferon beta (IFNβ) and Toll-like receptor 7 (TLR7) expression levels in the bursa increased significantly. This indicated that ALV-J stimulates a Type I IFN response. Next, we found that different ALV subgroups or strains up-regulated chicken IFN regulatory factor 3 (ChIRF-3) promoter activity, suggesting that ALV infection could trigger Type I IFNs pathway in vitro. Accordingly, we further investigated ChIFNα antiviral effects on ALV replication in DF-1 cells by successfully expressing recombinant ChIFNα in Escherichia coli (E. coli) strain BL21. The specific activity of the purified rChIFNα protein was determined to be 4 × 10⁷ U/mL. When added at 4000 U/mL, the recombinant protein restrained ALV replication as measured by decreases in viral protein p27 levels and mRNA expression. This new reagent may be useful for prophylactic and therapeutic drug design.     

A bioassay for interferon type I based on inhibition of Sendai virus growth

The expression of type I interferons (IFNs) in eukaryotic cells represents a first line of defense against viral infection. Cells pretreated by IFNs do not support viral replication and are protected from virus-induced cell destruction. A challenge of IFN-pretreated cells with vesicular stomatitis virus (VSV) is frequently used to quantitate this cytokine because, on the one hand, the replication of VSV is highly sensitive to IFNs and, on the other hand, in unprotected cells this virus induces a rapid cytopathic effect that can readily be quantified. However, as VSV may infect humans and is known to cause severe disease in a variety of animal species, this virus must be considered a biohazard. In this paper, we describe a bioassay for bovine IFN using Sendai virus, a paramyxovirus that grows readily in MDBK cells yet is released from these cells in a non-infectious form. The sensitivity and dynamic range of this assay are similar to those of the popular VSV-based IFN assay. We demonstrate that the Sendai-virus-based IFN assay permits rapid quantitation of recombinant bovine type I IFN, and also of native type I IFNs which are present in the supernatants of monocyte-derived macrophages infected with various pathogens. In view of the possible artifacts induced by viruses in samples to be assayed for IFN activity, we evaluated several methods of virus inactivation. Treatment with beta-propiolactone led to virus inactivation without affecting the bioactivity of IFNs as detected in the Sendai-virus-based assay.     

Subtypes of type I IFN differentially enhance cytokine expression by suboptimally stimulated CD4+ T cells

Human type I interferons (IFNs) include IFN‐β and 12 subtypes of IFN‐α. During viral infection, infiltrating memory CD4 ⁺ T cells are exposed to IFNs, but their impact on memory T‐cell function is poorly understood. To address this, we pretreated PBMCs with different IFNs for 16 h before stimulation with Staphylococcus aureus enterotoxin B and measured cytokine expression by flow cytometry. IFN‐α8 and ‐α10 most potently enhanced expression of IFN‐γ, IL‐2, and IL‐4. Potency among the subtypes differed most at doses between 10 and 100 U/mL. While enhancement of IL‐2 and IL‐4 correlated with the time of preincubation with type I IFN, IFN‐γ production was enhanced best when IFN‐α was added immediately preceding or simultaneously with T‐cell stimulation. Comparison of T‐cell responses to multiple doses of Staphylococcus aureus enterotoxin B and to peptide libraries from RSV or CMV demonstrated that IFN‐α best enhanced cytokine expression when CD4 ⁺ T cells were suboptimally stimulated. We conclude that type I IFNs enhance Th1 and Th2 function with dose dependency and subtype specificity, and best when T‐cell stimulation is suboptimal. While type I IFNs may beneficially enhance CD4 ⁺ T‐cell memory responses to vaccines or viral pathogens, they may also enhance the function of resident Th2 cells and exacerbate allergic inflammation.     

Lack of a role for type I and type II interferons in the resolution of rotavirus-induced diarrhea and infection in mice.

Rotavirus infects the intestinal epithelium of most mammalian species and causes diarrhea in infants. Previously, we have shown that both type I and II human interferons (IFNs) have potent and mechanistically discreet antiviral effects in vitro against rotavirus. We have also shown that adult IFN-gamma knockout (-/-) mice have no alteration in clearance of primary rotavirus infection. In the present studies, we wished to determine the importance of both IFN types in modulation of degree and duration of disease and infection in mice. Immunocompetent suckling mice were treated orally (5,000 IU) or parenterally (500 IU) with type I and II murine IFNs before and after challenge with virulent murine rotavirus. Treated animals developed diarrhea indistinguishable from that observed in untreated control mice. In other experiments, type I IFN receptor -/- suckling mice and IFN-gamma-/- suckling mice developed diarrhea of similar characteristics and duration and had comparable quantities of viral antigen in their intestines as did immunocompetent mice. Furthermore, type I IFN receptor -/- adult mice infected with rotavirus shed equivalent quantities of viral antigen and with similar kinetics as the control mice. Thus, IFNs do not seem to be major inhibitors of rotavirus diarrhea or replication in mice.     

Autoimmunity is a type I interferon-deficiency syndrome corrected by ingested type I IFN via the GALT system.

Type I interferons (IFN-alpha/beta), products of the innate immune system, can modulate immune function whereas proinflammatory IFN-gamma (type II IFN), a product of the acquired immune system upregulates inflammation and enhances cell mediated immunity. We have proposed a unifying hypothesis of the origin of autoimmunity as a type I IFN immunodeficiency syndrome involving inadequate regulation of the acquired immune system product IFN-gamma by the IFN-alpha/beta innate immune system. The common theme of ingested type I IFNs in autoimmunity is inhibition of proinflammatory type II IFN systemically or at the target organ. In multiple sclerosis (MS) and insulin-dependent diabetes mellitus (IDDM) at the target organ, and in rheumatoid arthritis (RA) as a regulator of other proinflammatory cytokines, IFN-gamma is the nexus of inflammation in autoimmunity. Ingested type I IFNs counteract type II IFN, overcome the relative lack of type I IFN activity, and ameliorate autoimmunity. The administration of type I IFNs (IFN-alpha/beta) via the gut offers an exciting alternative to systemic application for overcoming the type I IFN immunodeficiency in autoimmunity. Successful use of ingested type I IFN in three separate prototypical autoimmune diseases suggests a broad antiinflammatory therapeutic profile for this technology.     

Enterovirus 71 blocks selectively type I interferon production through the 3C viral protein in mice

Type I interferons (IFNs) represent an essential innate defense mechanism for controlling enterovirus 71 (EV 71) infection. Mice inoculated with EV 71 produced a significantly lower amount of type I IFNs than those inoculated with poly (I:C), adenovirus type V, or coxsackievirus B3 (CB3). EV 71 infection, however, mounted a proinflammatory response with a significant increase in the levels of serum and brain interleukin (IL)‐6, monocyte chemoattractant protein‐1, tumor necrosis factor, and IFN‐γ. EV 71 infection abolished both poly (I:C)‐ and CB3‐induced type I IFN production of mice. Such effect was not extended to other enteroviruses including coxsackievirus A24, B2, B3, and echovirus 9, as mice infected with these viruses retained type I IFN responsiveness upon poly (I:C) challenge. In addition, EV 71‐infected RAW264.7 cells produced significantly lower amount of type I IFNs than non‐infected cells upon poly (I:C) stimulation. The inhibitory effect of EV 71 on type I IFN production was attributed to the viral protein 3C, which was confirmed using over‐expression systems in both mice and RAW264.7 cells. The 3C over‐expression, however, did not interfere with poly (I:C)‐induced proinflammatory cytokine production. These findings indicate that EV 71 can hamper the host innate defense by blocking selectively type I IFN synthesis through the 3C viral protein. J. Med. Virol. 84:1779–1789, 2012. © 2012 Wiley Periodicals, Inc.     

Efficacy of low-dose oral use of type I interferon in cytomegalovirus infections in vivo.

Oral administration of type I interferons (IFNs; murine IFN-alpha and IFN-beta) reduces early replication of murine cytomegalovirus (MCMV) in both the spleen and liver of MCMV-infected BALB/c mice. Examination of a range of doses of IFN (1 to 1000 IU) showed that 10 IU administered daily for 1 week prior to virus infection was optimal for inhibition of MCMV replication. Furthermore, low-dose orally administered IFN (10 IU/day) was effective in mice challenged with lethal and sublethal virus inocula. The antiviral efficacy of low-dose orally administered IFN was not restricted by either the route of virus inoculation or the mouse genotype. Analysis by immunohistochemistry of IFN-alpha receptor-bearing cells of the gastrointestinal tract revealed predominant staining of perivascular smooth muscle and the lamina propria of the anterior tongue, small intestine and rectum. These tissues, dense in IFN-alpha receptor-bearing cells, are likely to be the sites of interaction of the orally administered IFNs with the mucosal immune system. In conclusion, we propose that low-dose oral use of type I IFN therapy may have broad applications in the treatment of CMV infections.     

Plasmacytoid dendritic cell precursors/type I interferon-producing cells sense viral infection by Toll-like receptor (TLR) 7 and TLR9

Plasmacytoid dendritic cell (pDC) precursors, also called type I IFN (//)-producing cells (IPCs), are the key effectors in the innate immune system because of their extraordinary capacity to produce type I IFNs against microbial infection, particularly viral infection. In contrast to myeloid DCs, human pDC/IPCs selectively express Toll-like receptor (TLR) 7 and TLR9 within the endosomal compartment. These receptors are specifically designed to recognize the nucleoside-based products derived from RNA viruses and DNA viruses. Therefore, this expression profile potentially enables pDC/IPCs to sense a variety of viruses. Stimulation of TLR7 or TLR9 leads to type I IFN responses through the MyD88 pathway. Thus, pDC/IPCs may play a central role in host defense against viral infection through the TLR7 and TLR9 system.     

Multi-Step Regulation of Interferon Induction by Hepatitis C Virus

Acute hepatitis C virus (HCV) infection evokes several distinct innate immune responses in host, but the virus usually propagates by circumventing these responses. Although a replication intermediate double-stranded RNA is produced in infected cells, type I interferon (IFN) induction and immediate cell death are largely blocked in infected cells. In vitro studies suggested that type I and III IFNs are mainly produced in HCV-infected hepatocytes if the MAVS pathway is functional, and dysfunction of this pathway may lead to cellular permissiveness to HCV replication and production. Cellular immunity, including natural killer cell activation and antigen-specific CD8 T-cell proliferation, occurs following innate immune activation in response to HCV, but is often ineffective for eradication of HCV. Constitutive dsRNA stimulation differs in output from type I IFN therapy, which has been an authentic therapy for patients with HCV. Host innate immune responses to HCV RNA/proteins may be associated with progressive hepatic fibrosis and carcinogenesis once persistent HCV infection is established in opposition to the IFN system. Hence, innate RNA sensing exerts pivotal functions against HCV genome replication and host pathogenesis through modulation of the IFN system. Molecules participating in the RIG-I and Toll-like receptor 3 pathways are the main targets for HCV, disabling the anti-viral functions of these IFN-inducing molecules. We discuss the mechanisms that abolish type I and type III IFN production in HCV-infected cells, which may contribute to understanding the mechanism of virus persistence and resistance to the IFN therapy.     

Expression of type III interferons (IFNλs) and their receptor in Sjögren's syndrome

Type III interferons (IFNs) or IFN‐λs (IFN‐λ1/IL29, IFN‐λ2/interleukin (IL)?28A and IFN‐λ3/IL‐28B) consist of a recently identified group of IFNs, implicated initially in several human diseases, including cancer and autoimmunity. In this study, we sought to investigate the expression of type III IFNs and their common receptor IFN‐λR1/IL‐28Ra in Sjögren's syndrome (SS). Type III IFN expression was examined in minor salivary gland tissues (MSG), peripheral blood mononuclear cells (PBMCs), sera and resting or Toll‐like receptor (TLR)‐stimulated salivary gland epithelial cells (SGEC) from SS patients and sicca‐complaining controls. All type III IFN family members were detected in ductal and acinar epithelia of MSGs from both SS patients and sicca controls. IFN‐λ2/IL‐28A and IFN‐λ3/IL‐28B were also expressed in infiltrating mononuclear cells. In SS patients with intermediate MSG lesions, the epithelial expression of IFN‐λ2/IL‐28A was more intense compared to sicca controls (P < 0·05). The receptor IFN‐λR1/IL‐28Ra was detected in all types of cells except fibroblasts, and was exceptionally strong in plasmatocytoid dendritic cells, indicating that they are susceptible to type III IFN‐mediated regulation. In the periphery, only IFN‐λ1/IL‐29 was detected in the sera and was elevated significantly in SS patients with intermediate MSG inflammatory lesions compared to sicca controls (P = 0·0053). None of the type III IFNs was expressed constitutively in resting SGECs; they were all induced readily by TLR‐3 stimulation, suggesting that the in‐situ epithelial expression can be attributed to local microenvironment. Type III IFNs are expressed in MSGs in a similar pattern to type I IFNs and their expression is probably subjected to micro‐environmental regulation, suggesting that they are implicated in the inflammatory processes occurring in the affected exocrine glands.