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 expression induced by α-defensin in healthy human periodontal tissue

Although periodontal tissue is continually challenged by microbial plaque, it is generally maintained in a healthy state. To understand the basis for this, we investigated innate antiviral immunity in human periodontal tissue. The expression of mRNA encoding different antiviral proteins, myxovirus resistance A (MxA), protein kinase R (PKR), oligoadenylate synthetase (OAS), and secretory leukocyte protease inhibitor (SLPI) were detected in both healthy tissue and that with periodontitis. Immunostaining data consistently showed higher MxA protein expression in the epithelial layer of healthy gingiva as compared with tissue with periodontitis. Human MxA is thought to be induced by type I and III interferons (IFNs) but neither cytokine type was detected in healthy periodontal tissues. Treatment in vitro of primary human gingival epithelial cells (HGECs) with α-defensins, but not with the antimicrobial peptides β-defensins or LL-37, led to MxA protein expression. α-defensin was also detected in healthy periodontal tissue. In addition, MxA in α-defensin-treated HGECs was associated with protection against avian influenza H5N1 infection and silencing of the MxA gene using MxA-targeted-siRNA abolished this antiviral activity. To our knowledge, this is the first study to uncover a novel pathway of human MxA induction, which is initiated by an endogenous antimicrobial peptide, namely α-defensin. This pathway may play an important role in the first line of antiviral defense in periodontal tissue.     

Enhanced type I interferon signalling promotes Th1-biased inflammation in cutaneous lupus erythematosus

Recent studies have suggested that type I interferons (IFN) play a role in the pathogenesis of lupus erythematosus (LE), an autoimmune disease of unknown aetiology. Natural interferon-producing plasmacytoid cells have been demonstrated in cutaneous LE (CLE) lesions, along with elevated levels of IFN-alpha mRNA. The hypothesis in the current study was that local production of type I IFNs in CLE induces Th1-biased inflammation via induction of IFN-inducible chemokines such as IP10/CXCL10 leading to the recruitment of chemokine receptor CXCR3 expressing T-cells into skin lesions. Skin biopsies from 21 patients suffering from different types of active cutaneous LE were analysed for the expression of MxA, a protein specifically induced by type I interferons, the IFN-inducible protein IP10/CXCL10, and the chemokine receptor CXCR3, characteristic for Th1 cells, by immunohistochemistry. Additionally, peripheral CD4+ and CD8+ T-cells were investigated for the expression of MxA and CXCR3 by flow cytometry. Cutaneous LE lesions were characterized by strong expression of MxA indicating the induction of localized type I IFN signalling in the skin. Large numbers of infiltrating CXCR3 positive lymphocytes were detected in CLE skin lesions, and correlated closely with lesional MxA expression (epidermis: Spearman's rho = 0.56, p < 0.001; dermis: rho = 0.82, p < 0.001). Intracellular MxA levels of circulating CD4+ and CD8+ T-cells were significantly enhanced in patients with active CLE lesions. The percentage of peripheral T-cells expressing CXCR3 was significantly decreased in specific CLE subtypes. Expression of IP10/CXCL10 in the epidermis links type I IFN signalling and recruitment of CXCR3+ T cells. These results suggest an important role for type I interferon signalling in the pathogenesis of cutaneous lupus erythematosus. It is proposed that type I IFNs induce a Th1-biased inflammatory immune response, with recruitment of CXCR3-expressing T-lymphocytes into the skin.     

腺病毒3型诱导MxA蛋白产生及其抗病毒作用的研究

目的:观察腺病毒3型对MxA蛋白的诱导作用以及MxA蛋白的体外抗病毒作用。方法:采用流式细胞仪分析不同浓度的腺病毒3型（Ad3）诱导健康人外周血单个核细胞（PBMC）胞浆MxA蛋白的产生;采用微量细胞病变抑制法观察重组MxA蛋白对Hela细胞内腺病毒3型的抗病毒作用。结果:不同浓度的腺病毒诱异PBMC产生MxA蛋白的含量均显著高于对照组。10 ng/ml MxA蛋白质抗Hela细胞内腺病毒3型感染的效价为20TCID20。结论:腺病毒3型体外可诱导PBMC产生MxA蛋白;重组MxA蛋白具有抗腺病毒3型作用。     

Difference in susceptibility to MxA protein between a coxsackievirus B1 isolate and prototype,impact of serial cell culture passage

Human enteroviruses (EVs) cause a broad spectrum of acute and chronic diseases including meningitis and myocarditis. The type I interferon‐induced MxA protein has been shown to inhibit the replication of an EV, coxsackievirus B4 (CVB4), but not cardioviruses such as encephalomyocarditis virus and mengo virus, members of the Picornaviridae family. EVs consist of more than 60 distinct serotypes against which the antiviral activity of MxA was not investigated yet. The main aim of this study was to explore the antiviral activity of MxA protein against a clinical CVB1 isolate and other EV prototypes. Vero cells expressing constituvely MxA protein were infected with EVs, and the percentage of inhibiton of expression of enteroviral RNA and capsid VP1 protein was determined. Following infection of MxA‐transfected Vero cells with EVs, the expression of enteroviral RNA was inhibited by up to 99%, and that of VP1 protein by up to 85%. However, there was a difference in the percentage of MxA inhibition of EV replication between the different EV prototypes. This difference in MxA sensitivity was not due to a difference in the viral replication rates. The MxA protein was inactive against the clinical CVB1 isolate, and the replication rate of CVB1 isolate in MxA‐transfected Vero cells was higher than that in mock‐transfected Vero cells. A serial passage of the clinical CVB1 isolate and other EV prototypes resulted in an increase in their susceptibility to MxA protein. These results suggest the presence of MxA‐resistant EV variants that may escape innate immunity and cause disease. J. Med. Virol. 82:424–432, 2010. © 2010 Wiley‐Liss, Inc.     

Alpha interferon, antiviral proteins and their value in clinical medicine

Type I interferon system is an important part of host's innate defense mechanisms against viral infections. The type I interferons mediate in part their antiviral effect via induction of various proteins. Among them the most widely known are 2'-5' oligoadenylate synthetase (2'-5' OAS) and a protein kinase (PKR). MxA, an other antiviral protein, is specifically induced by the type I interferons. The MxA protein contains the dynamin signature, which is implicated in transport processes. The MxA protein appears to block the replication of certain viruses at poorly defined steps. There are substantial differences in the antiviral activity of MxA between virus types. Indeed, the replication of vesicular stomatitis virus and influenza virus is inhibited by MxA, but not the one of type I herpes simplex virus. Measurements of interferon alpha and MxA levels may be of high value in clinical practice. Interferon alpha can be detected by using a bioassay based on the interferon alpha ability to protect cultured cells from the cytopathic effect caused by a selected challenged virus, or by using immunological techniques. The current bioassays are the most sensitive methods but they are cumbersome and lengthy, even though simplifications have been proposed. Immunological techniques are easier, however they do not explore the biological activity of the circulating interferon. The presence of type I interferon in biological samples (serum, plasma, cerebro-spinal fluid, cultured cell supernatants) can be indirectly assessed by capability of interferon alpha to induce in vitro the synthesis of MxA in a dose dependent manner in cultured cells. Following to the lysis of the cells, the induced MxA can be quantitated and hence the type I-interferon concentration can be determinated in samples. The quantitation of MxA protein in peripheral blood lysates can be useful as a specific marker of acute viral infections. A minute amount of whole blood (15 mul) is sufficient which facilitates its use in pediatrics. The specifically type-I-interferons inducible MxA protein is also a potential useful marker in the management of interferon alpha-treatment. Moreover, the detection of interferon alpha and antiviral proteins constitute an indirect approach for investigating the hypothesis of the role of viruses in chronic diseases with suspected infectious aetiology.     

Interferon-inducible Mx gene expression in cotton rats: cloning, characterization, and expression during influenza viral infection.

Mx proteins belong to the superfamily of large GTPases with antiviral activity against a wide range of RNA viruses. In vivo, the expression of Mx genes is tightly regulated by the presence of type I interferons (IFNs), and their induction has been described during several viral infections. However, because of the absence of functional Mx genes in most common laboratory strains of mice, in vivo studies of the expression of these genes during viral infection have been hampered. We have cloned the cDNAs for the cotton rat homologs of Mx1 and Mx2 genes that encode full-length proteins. Mx1 localized in the nucleus, whereas Mx2, as its human homolog MxA, localized in the cytoplasm. The expression of Mx genes in cotton rat cells was induced by type I IFNs (IFN-alpha and IFN-beta) but induced only marginally with type II IFN (IFN-gamma). In vivo, the expression of Mx genes was dramatically augmented in lungs of cotton rats infected with influenza virus. The expression of Mx genes and protein(s) was dependent on the dose of virus and the time postinfection for the analysis. Our data present for the first time a complete analysis of the kinetics of expression of these influenza resistant genes in vivo and underscore the fidelity and sensitivity of the cotton rat model for the study of influenza viral infection.     

Global transcriptional profiling demonstrates the combination of type I and type II interferon enhances antiviral and immune responses at clinically relevant doses.

A role for type II interferon (IFN-gamma) in resolving viral infection is suggested by the correlation of hepatitis C virus (HCV) clearance with enhancement of IFN-gamma-producing activated T cells in the resolution of acute HCV infection. Using vesicular stomatitis virus (VSV), a synergistic direct antiviral effect was documented using IFN-gamma1b and a potent, consensus type I IFN (IFN alfacon-1). Global expression profiling following EC50 exposure to IFN alfacon-1, IFN-gamma1b, or a cocktail of the two allowed the antiviral state to be correlated with induction of a subset of IFN-stimulated genes (ISGs). Genes identified through this analysis corresponded to classic antiviral components, ISGs more recently associated with direct antiviral functions, as well as expressed sequence tags (ESTs) and hypothetical proteins. The magnitude of these antiviral EC50-correlated expression events in human hepatoma (Huh7) cells exposed to clinically relevant doses of IFN alfacon-1, IFN-gamma1b, or a cocktail of the two was also probed because the standard of care for patients with chronic hepatitis C is type I IFN-containing regimens. Relative to type I IFNs used alone, the addition of type II IFN caused enhanced expression not only of many of the genes correlated with the direct antiviral state but also of genes involved in (1) antigen presentation to cytotoxic T lymphocytes (CTLs), (2) macrophage, natural killer (NK), and T helper 1 (Th1) cell recruitment and activation, (3) complement system function, (4) apoptosis, and (5) ISGs with unknown functions. As many of these processes are correlated clinically with resolution of chronic HCV infection, the combined use of these IFNs could display a beneficial effect on viral clearance in patients infected with HCV and other viruses through enhancement of one of these processes or of the direct antiviral state.     

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.     

Viruses and the type I interferon antiviral system: induction and evasion

The type I interferon (IFN) system responds to viral infection and induces an "antiviral state" in cells, providing an important first line of defense against virus infection. Interaction of type I IFNs (IFN alpha and IFN beta) with their receptor induces hundreds of cellular genes. Of the proteins induced by IFN, the antiviral function of only a few is known, and their mechanisms of action are only partly understood. Additionally, although viral-encoded mechanisms that counteract specific components of the type I IFN response have been known for some time, it has recently become clear that many (if not most) viruses encode some form of IFN-antagonist. Understanding the interplay between viral-encoded IFN antagonists and the interferon response will be essential if the therapeutic potential of IFNs is to be fully exploited.     

Type I interferon-associated recruitment of cytotoxic lymphocytes: a common mechanism in regressive melanocytic lesions

We studied 253 primary melanomas of the skin for histologic signs of regression. Detailed immunohistologic analyses, including expression of MxA (an antiviral protein specifically induced by type I interferons), the chemokine IP10/CXCL10, the chemokine receptor CXCR3, and the cytotoxic molecule granzyme B, were performed for 14 typical regressive tumors and 20 control samples (congenital nevi, halo nevi, unaffected skin). We found high expression of MxA, indicating local type I interferon production, in inflamed regressive melanocytic lesions, along with large numbers of natural interferon-producing plasmacytoid dendritic cells, CXCR3+ lymphocytes, and granzyme B+ lymphocytes. We also detected high expression of the interferon-induced chemokine IP10/CXCL10, linking type I interferon production and recruitment of CXCR3+ lymphocytes. Our results provide evidence that endogenous activation of type I interferons, infiltration of plasmacytoid dendritic cells, and recruitment of CXCR3+ and granzyme B+ lymphocytes are involved in spontaneous regression of melanoma and other melanocytic lesions. We believe this cytotoxic immune response represents an evolutionarily conserved pathway against intracellular pathogens.     

IFN-λs

For decades, type I IFNs have been considered indispensable and unique antiviral mediators for the activation of rapid innate antiviral protection. However, the recent discovery of type III IFNs is challenging this paradigm. Since their identification in 2002/2003 by two independent groups, type III IFNs or IFN-λs, also known as IL-28/29, have been the subject of increased study with consequent recognition of their importance in virology and immunology. Initial reports suggested that IFN-λs functionally resemble type I IFNs. Although IFN-λs and classical type I IFNs (IFN-α/β) utilize distinct receptor complexes for signaling, both types of IFNs activate similar intracellular signaling pathways and biological activities, including the ability to induce antiviral state in cells, and both type I and type III IFNs are induced by viral infection. However, different antiviral potency, pattern of their induction and differential tissue expression of their corresponding receptor subunits suggest that the type I and type III IFN antiviral systems do not merely duplicate each other. Recent studies have started to reveal unique biological activities of IFN-λs in and beyond innate antiviral immunity.     

Interferon-stimulated genes response in endothelial cells following Hantaan virus infection

The regulation mechanism of interferon (IFN) and IFN-stimulated genes is a very complex procedure and is dependent on cell types and virus species. We observed molecular changes related to anti-viral responses in endothelial cells during Hantaan virus (HTNV) infection. We found that there are two patterns of gene expression, the first pattern of gene expression being characterized by early induction and short action, as in that of type I IFNs,' and the other being characterized by delayed induction and long duration, as those of IRF-7, MxA, and TAP-1/2. Even though there are significant differences in their induction folds, we found that all of IFN-alpha/beta, IRF- 3/7, MxA, and TAP-1/2 mRNA expressions reached the peak when the viral replication was most active, which took place 3 days of post infection (d.p.i.). In addition, an interesting phenomenon was observed; only one gene was highly expressed in paired genes such as IFN-alpha/beta' (3/277-folds), IRF-3/7 (2.2/29.4-folds), and TAP- 1/2 (26.2/6.1-folds). Therefore, IFN-beta, IRF-7, and TAP-1 seem to be more important for the anti-viral response in HTNV infection. MxA was increased to 296-folds at 3 d.p.i. and kept continuing 207-folds until 7 d.p.i.. The above results indicate that IFN-beta works for an early anti-viral response, while IRF7, MxA, and TAP-1 work for prolonged anti-viral response in HTNV infection.     

Inhibition of coxsackievirus B4 replication in stably transfected cells expressing human MxA protein

Coxsackieviruses B (CVB) (B1-B6), positive-strand RNA viruses, cause a variety of diseases. CVB4 may have a causal role in insulin-dependent diabetes mellitus. IFN-alpha inhibits CVB replication; however, the mechanism is not well known. The interferon-alpha-inducible human MxA protein exerts an antiviral activity against negative-strand RNA viruses and against Semliki Forest virus, a positive-strand RNA virus. To test the antiviral spectrum of MxA against CVB4, we took advantage of stably transfected Vero cells expressing MxA (Vero/MxA) in 98% of cells. Compared with control cells, in Vero/MxA cells, CVB4 yields were dramatically reduced and expression of the VP1 CVB protein analyzed by immunofluorescence was highly restricted. Furthermore, the accumulation of positive- and negative-strand CVB4 RNA was prevented as shown by in situ hybridization and RT-PCR. These results indicate that the antiviral activity of MxA extends to CVB4 and that its replication cycle is inhibited at an early step in Vero/MxA cells.