The biochemical mechanisms of action of the interferons

The interferon family of proteins consists of a variety of antigenically distinct types that are encoded by different genes. Several of these genes have been cloned and the availability of large amounts of the recombinant interferons has greatly facilitated characterization of their biochemical and biological activities. The specificity of interaction of interferons with cell surface receptors has been investigated in binding and covalent cross-linking experiments employing 125I-labeled interferons. In addition to their antiviral activity, the interferons have effects on cell growth and differentiation. The interferons also are potent modulators of the immune response. The interferons exert their biological activities by altering the expression of several cellular proteins. Current knowledge about the mechanisms of signal transduction and the regulation of expression of interferon-induced mRNAs and proteins are discussed.     

An Image-Based Genetic Assay Identifies Genes in T1D Susceptibility Loci Controlling Cellular Antiviral Immunity in Mouse

The pathogenesis of complex diseases, such as type 1 diabetes (T1D), derives from interactions between host genetics and environmental factors. Previous studies have suggested that viral infection plays a significant role in initiation of T1D in genetically predisposed individuals. T1D susceptibility loci may therefore be enriched in previously uncharacterized genes functioning in antiviral defense pathways. To identify genes involved in antiviral immunity, we performed an image-based high-throughput genetic screen using short hairpin RNAs (shRNAs) against 161 genes within T1D susceptibility loci. RAW 264.7 cells transduced with shRNAs were infected with GFP-expressing herpes simplex virus type 1 (HSV-1) and fluorescent microscopy was performed to assess the viral infectivity by fluorescence reporter activity. Of the 14 candidates identified with high confidence, two candidates were selected for further investigation, Il27 and Tagap. Administration of recombinant IL-27 during viral infection was found to act synergistically with interferon gamma (IFN-γ) to activate expression of type I IFNs and proinflammatory cytokines, and to enhance the activities of interferon regulatory factor 3 (IRF3). Consistent with a role in antiviral immunity, Tagap-deficient macrophages demonstrated increased viral replication, reduced expression of proinflammatory chemokines and cytokines, and decreased production of IFN-β. Taken together, our unbiased loss-of-function genetic screen identifies genes that play a role in host antiviral immunity and delineates roles for IL-27 and Tagap in the production of antiviral cytokines.     

Ⅲ型干扰素——新型抗病毒细胞因子

干扰素（IFN）是抗病毒感染的第一道防线,Ⅰ型和Ⅱ型干扰素不仅可抑制病毒,而且还能参与天然免疫反应和获得性免疫反应。最近干扰素家族增添一位新成员：Ⅲ型干扰素,即IFN-λ,因其具有类似干扰素的抗病毒活性且能诱导干扰素相关基因的表达而命名。IFN-λ受体与Ⅰ型干扰素的受体不同,但具有与Ⅰ型干扰素类似的诱导表达方式和信号转导通路,并能激活一系列相似的干扰素刺激基因。就IFN-λ家族及其受体、基因表达和信号转导机制、抗病毒作用等进行综述。     

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.     

Purification techniques for human interferons

This article outlines the development and general status of present purification methods for human interferons. The isolation of each interferon species from natural sources is extremely difficult because of molecular characteristics, high losses of activity and the small amount of interferon protein present in production media. Few of the highly sophisticated methods meet the requirements for scale-up or give acceptable recoveries for a production process. The adaptation of purification procedures to the different interferon species is described, such as initial concentration, the extraction of beta interferon (IFN-β) by aqueous two-phase systems and the final purification of alpha interferon (IFN-α) and beta interferon to homogeneity. H.p.l.c. techniques are discussed in more detail together with problems in the purification of beta interferon and gamma interferon (IFN-γ). The range of interferon expression and excretion in recombinant microbial and animal cells is reviewed and different approaches for the solubilization and purification of intracellular recombinant interferons are described, which are covered mainly in patent applications.     

Antiviral activity of mutant interferons. A new approach to the study of structural-functional organization of interferons

The developed approach to investing the structure-functional organization of interferon has been developed consisting in: 1) fusing genes of interferon and alpha-peptide of beta-galactosidase, the resultant protein having the interferon properties and being determined by the beta-galactosidase alpha-complementation test; 2) constructing mutant genes of interferon by the localized mutagenesis; 3) determining the mutant interferon activity; 4) deducing the amino acid sequence of mutant interferon by sequencing mutant genes; 5) analyzing structure-functional organization of interferon. In accordance with this approach, ten mutant interferons with up to 15 changes of amino acid substitutions are obtained and their antiviral activity is determined. The role of some amino acid residues in antiviral activity of interferon alpha 2 is revealed.     

Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.     

Relative sensitivity of hepatitis B virus and other hepatotropic viruses to the antiviral effects of cytokines

We have previously shown that hepatitis B virus (HBV) replication is inhibited noncytopathically in the livers of transgenic mice following injection of HBV-specific cytotoxic T lymphocytes (CTLs) or infection with unrelated hepatotropic viruses, including lymphocytic choriomeningitis virus (LCMV) and adenovirus. These effects are mediated by gamma interferon (IFNgamma), tumor necrosis factor alpha (TNFalpha), and IFNalpha/beta. In the present study, we crossed HBV transgenic mice with mice genetically deficient for IFNgamma (IFNgammaKO), the TNFalpha receptor (TNFalphaRKO), or the IFNalpha/beta receptor (IFNalpha/betaRKO) in order to determine the relative contribution of each cytokine to the antiviral effects observed in each of these systems. Interestingly, we showed that HBV replicates in unmanipulated IFNgammaKO and IFNalpha/betaRKO mice at levels higher than those observed in control mice, implying that baseline levels of these cytokines control HBV replication in the absence of inflammation. We also showed that IFNgamma mediates most of the antiviral effect of the CTLs while IFNalpha/beta is primarily responsible for the early inhibitory effect of LCMV and adenovirus on HBV replication. In addition, we showed that the hepatic induction of IFNalpha/beta observed after injection of poly(I. C) is sufficient to inhibit HBV replication and that a similar antiviral effect is achieved by systemic administration of very high doses of IFNalpha. We also compared the relative sensitivity of LCMV and adenovirus to control by IFNgamma, TNFalpha, or IFNalpha/beta in these animals. Importantly, IFNalpha/betaRKO mice, and to a lesser extent IFNgammaKO mice, showed higher hepatic levels of LCMV RNA and adenovirus DNA and RNA than control mice, underscoring the importance of both interferons in controlling these other viral infections as well.     

New interferons in the treatment of chronic hepatitis C

The current standard therapy for chronic HCV infection is a combination of pegylated-interferon (PEG-IFN) and weight-based ribavirin, administered for 24-48 weeks, according to the viral genotype. Although the weekly administration of pegylated interferons provides superior antiviral efficacy over standard interferon alpha, the rate of sustained virological response rarely overpasses 50% in patients infected with HCV genotypes 1 and 4. Consequently, multiple clinical trials with congeners of interferon (consensus interferon, interferon lambda, albinterferon, and controlled-release interferons) are ongoing. Their main advantages consist in maintenance of viral suppression across a longer dosing interval, avoidance of interdose trough and reduced dosing frequencies (twice or even once per month compared to once per week for the actual PEG-IFNs). Along with these superior pharmacokinetic properties, new interferons are expected to have improved side-effect profiles and better tolerability compared with the currently available formulations, providing an option for otherwise difficult to treat, challenging populations. New interferon formulation can be incorporated into future combination with direct acting antivirals, in order to maintain viral suppression over longer periods and minimize the development of viral resistance.     

Comparative analysis of anti-hepatitis C virus activity and gene expression mediated by alpha,beta, and gamma interferons

A direct comparison of the inhibitory effects of alpha, beta, and gamma interferons (IFNs) on replication of a hepatitis C virus subgenomic replicon in a hepatoma cell line revealed similarities in antiviral potency. However, alternate IFN-induced antiviral mechanisms were suggested following observations of striking differences between IFN-gamma and IFN-alpha/beta with respect to strength and durability of the antiviral response and the magnitude and pattern of IFN-mediated gene expression.     

Some biological properties of the human amniotic membrane interferon.

Human amniotic interferon was investigated to define the species specificity of its antiviral action and to compare its anti-cellular and NK cell stimulating activities with those of other human interferons. The antiviral effect was titrated in bovine (RV-IAL) and monkey (VERO) cells. Amniotic interferon exhibited, in bovine cells, 5% of the activity seen in monkey cells, while alpha interferon displayed 200%. No effect was detected with either beta or gamma interferon in bovine cells. Daudi cells were exposed to different concentrations of various interferons and the cell numbers were determined. The anticellular effect of the amniotic interferon reached its peak on the third day of incubation. Results suggested a higher activity for alpha and gamma interferons and a lower activity for beta when compared to amniotic interferon. Using total mononuclear cells as effector cells and K 562 as target cells in a 51Cr release assay, it was demonstrated that low concentrations of amniotic interferon consistently stimulated NK cell activity in cells derived from several donors, the results indicating a higher level of activity with this interferon than with alpha and beta interferons.     

Immuno-modulating properties of interferons

Interferons (IFNs), by inducing the antiviral state in cells, are in the first line of defence against virus infections and are therefore part of the immune system as defined in its broadest sense. In addition, IFN-alpha and beta can influence specific functions of lymphocytes and macrophages; moreover, a special class of interferons, called IFN-gamma, are produced as a result of antigen recognition by T cells and by the interaction of mitogens and lymphocytes. Interferons influence B and T cell function in vitro and in vivo, as demonstrated by their effects on antibody formation, specific cytotoxicity of sensitized T cells, allograft survival, delayed-type hypersensitivity and graft-host reaction. They stimulate Natural Killer cell activity and induce functional and morphological changes in mononuclear phagocytes. There are many examples of disturbed immune reactions as a result of viral infection, implicating interferons as contributing factors; this is a result of the lack of immunological specificity of interferon action. The extent to which interferons have a truly immunoregulatory role is a question currently receiving a great deal of attention, but is still very much unsettled.     

Relationship of cellular oncogene expression to inhibition of growth and induction of differentiation of Daudi cells by interferons or TPA

Human alpha or beta interferons inhibit the proliferation of Daudi Burkitt lymphoma cells and induce the differentiation of these cells towards a mature plasma cell phenotype. Similar responses are seen when Daudi cells are treated with the phorbol ester, TPA. Both interferons and TPA down-regulate expression of the c-myc oncogene in these cells. Although TPA can mimic the effect of interferon on cell differentiation, it does not induce 2'5' oligoadenylate synthetase or the interferon-sensitive mRNAs, 6-16 or 9-27. Thus chronic stimulation of protein kinase C by TPA cannot mimic all of the effects of interferon treatment on gene expression. Inhibition of ADP-ribosyl transferase activity by 3-methoxybenzamide impairs interferon- or TPA-induced differentiation of Daudi cells. This agent induces a higher level of c-myc mRNA in the cells and stimulates the incorporation of [3H]thymidine into DNA; although these effects are partially counteracted by interferon or TPA treatment, the elevated expression of the c-myc gene may be sufficient to prevent terminal differentiation and allow cell proliferation to continue.     

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma)

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.     

2’-5’寡聚腺苷酸合成酶（OAS）及其临床意义的研究进展

干扰素作用于靶细胞膜表面的受体后,通过信号转导系统诱导一系列抗病毒蛋白产生,干扰病毒复制以达到抗病毒目的。2’-5’寡聚腺苷酸合成酶（2’．5’oligoadenylatesynthetase,OAS）是干扰素作用于细胞后产生的一种重要的抗病毒蛋白,几十年来,国内外学者对OAS家族及其抗病毒机制进行了大量研究并取得了一定的进展,OAS被dsRNA激活后,催化生成2-5A,2-5A激活核酸内切酶RNaseL,降解病毒RNA,阻断病毒蛋白合成,从而发挥抗病毒作用。体内外研究表明,OAS的表达量或活性的变化可用于评价机体对干扰素的反应,反映干扰素抗病毒效果,另外,它还可作为系统性红斑狼疮的病情活动度的一种检测指标。因此,OAS具有重要的临床应用价值。本文就OAS家族及其抗病毒机制,其测定方法与对于病毒性肝炎和系统性红斑狼疮疾病的临床意义展开综述,以期对OAS的研究和应用提供参考。OAS是典型的干扰素诱导产物,可反映机体内干扰素的抗病毒水平,具有广阔的应用前景。