鱼类干扰素系统基因研究进展

干扰素是一类多基因家族诱导性细胞因子,可在脊椎动物细胞中诱导建立抗病毒状态,并在抗病毒防御中起重要作用。干扰素系统包括应答外界刺激(如病毒感染)而合成干扰素的细胞,和应答干扰素建立抗病毒状态的细胞。干扰素系统基因主要包括Ⅰ型干扰素、Ⅱ型干扰素、干扰素刺激基因以及组成干扰素信号传导系统的基因。鱼类干扰素类作用发现较早,而对其基因的克隆和鉴定较晚。随着哺乳类和鸟类中干扰素及其相关基因研究的开展和深入,近年来鱼类干扰素及其相关基因的研究也得到快速发展。综述了鱼类干扰素系统基因的克隆、鉴定,以及其功能方面的研究进展。     

鱼类培养细胞干扰素的诱导

对紫外线灭活的草鱼出血病病毒（ＧＣＨＶ）－Ｆ９株,在几种鲤科鱼类培养细胞中诱导产生干扰素的能力及影响干扰素产量的各因素进行了研究。纯化的ＧＣＨＶ在紫外线照射５分丧失感染性,但获得了在ＣＡＢ等５种鲤科鱼类培养细胞中高铲诱导产生干扰素的能力。干扰素的诱导需要病毒高复数感染细胞。干扰素主要在诱导后１４小时时内产生。培养上清中的新生牛血清对干扰素的产量有抑制作用。而在ＰＨ６．２－７．８范围内对干扰素产量无     

干扰素诱导的鱼类Mx蛋白

Mx蛋白是干扰素诱导表达的蛋白家族中的成员,当机体和细胞受病毒感染或诱生剂处理时产生。Mx蛋白和其它干扰素诱导蛋白一起构成宿主细胞的抗病毒状态,以达到抗病毒的目的。研究表明,Mx蛋白具有抗病毒活性,还可能与其它基本生命活动如发育或分化,蛋白质分送和生长有关。在鱼类也发现多种Mx蛋白,具有Mx蛋白家族的共有特征;在肽链末端有一个三联ATP／GTP结合区和发动蛋白家族的结构特征序列;在蛋白C端存在使Mx蛋白形成三聚体的Leu拉链结构以及定位信号。但是迄今没有发现鱼类Mx蛋白的抗病毒活性。文章最后对目前鱼类病毒病的防治及利用抗病毒基因进行鱼类基因工程抗病毒育种进行了探讨。     

干扰素的信号传导和抗病毒效应机制

干扰素是一种具有抗病毒、抗增殖和免疫调节功能的细胞因子,它在宿主天然免疫防御中起着重要的作用。干扰素诱导的信号通路除了最初的Jak-Stat途径以外,很多新发现的信号途径对于干扰素反应也是必需的。干扰素反应最终导致抗病毒的效应蛋白通路,包括2′,5′-寡聚腺苷酸合成酶、蛋白激酶、ISG15等途径。这些效应蛋白通过抑制病毒转录、降解病毒RNA、抑制翻译和修饰蛋白的功能来控制病毒复制的过程。     

干扰素诱导的鱼类Mx蛋白

Mx蛋白是干扰素诱导表达的蛋白家族中的成员,当机体和细胞受病毒感染或诱生剂处理时产生.Mx蛋白和其它干扰素诱导蛋白一起构成宿主细胞的抗病毒状态,以达到抗病毒的目的.研究表明,Mx蛋白具有抗病毒活性,还可能与其它基本生命活动如发育或分化,蛋白质分送和生长有关.在鱼类也发现多种Mx蛋白,具有Mx蛋白家族的共有特征在肽链末端有一个三联ATP/GTP结合区和发动蛋白家族的结构特征序列;在蛋白C端存在使Mx蛋白形成三聚体的Leu拉链结构以及定位信号.但是迄今没有发现鱼类Mx蛋白的抗病毒活性.文章最后对目前鱼类病毒病的防治及利用抗病毒基因进行鱼类基因工程抗病毒育种进行了探讨.     

泛素样蛋白ISG15在抗病毒天然免疫中的作用

干扰素刺激基因15(ISG15)编码的蛋白是抗病毒天然免疫通路中的重要调节因子,病毒感染和干扰素刺激均可强烈诱导ISG15的表达。ISG15是最早发现的泛素样蛋白,可对细胞内多种蛋白进行修饰并调节蛋白功能,但不介导蛋白质的降解,在机体抗病毒天然免疫反应中发挥重要作用,其机制尚未完全明确。近几年对ISG15的研究有所突破,发现了ISG15在抗病毒天然免疫反应中的新功能。我们简要概述了泛素样蛋白ISG15的概况、修饰酶系统及ISG15在抗病毒天然免疫反应中功能的研究进展。     

干扰素产品现状及市场前景

干扰素产品现状及市场前景吴美英程永庆（北京三元基因工程有限公司）干扰素系统是一种重要的细胞功能调节系统。干扰素具有广谱的抗病毒,抗细胞分裂,免疫调节等多种生物活性。它是一种重要的抗病毒、抗肿瘤治疗药物［１］。干扰素的种类很多,有人体干扰素,动物干扰素...     

鱼类和哺乳类RLR介导的抗病毒免疫反应的泛素化修饰调控

RLR[retinoic acid-inducible gene Ⅰ(RIG-Ⅰ)-like Receptors]是一类表达在胞浆中的模式识别受体, 在识别细胞质中经病毒复制产生的病毒RNA后, 启动一系列信号级联反应, 以诱导机体Ⅰ型干扰素及干扰素诱导的抗病毒基因的表达, 最后达到清除机体病毒感染的目的。由于在病毒感染时机体干扰素反应必须迅速启动, 当病毒清除后干扰素反应又需要立即恢复到正常本底水平, 因此RLR激活的信号转导途径受到了严格的调控, 其中就包括由E3泛素连接酶参与的泛素化修饰调控和由去泛素化酶参与的去泛素化修饰调控。自2003年成功鉴定出鱼类干扰素基因以来, 鱼类也被发现具有保守的RLR信号转导途径诱导干扰素抗病毒免疫反应, 该信号途径同样受到泛素化修饰的调控。文章总结了近年来泛素化修饰在哺乳类和鱼类RLR介导的抗病毒免疫应答通路中的调节机制。     

猪I型与II型干扰素的克隆、表达及抗病毒活性比较

干扰素(IFN)是由多种细胞受病毒感染或其他生物诱导剂刺激而产生的天然蛋白质,主要功能为抗病毒增殖、调节免疫反应和激活免疫细胞等。本研究克隆并测序了猪干扰素(PoIFN)α、γ、αγ及ω基因。构建原核表达载体pET-His/PoIFN-α、pET-His/PoIFN-γ、pET-His/PoIFN-αγ和pET-His/PoIFN-ω,转化大肠杆菌Rosetta(DE3)进行表达,经纯化、复性得到具有生物学活性的蛋白。用细胞病变抑制法在Marc-145/PRRSV、Marc-145/VSV、PK-15/VSV、Vero/VSV、MDBK/VSV系统上进行抗病毒活性测定,结果表明猪α和αγ融合干扰素有较为显著的抗病毒活性,抗PRRSV活性高达108U/mg;猪γ干扰素活性效价约为α干扰素的1/2到1/3;猪ω干扰素几乎未检测到抗病毒活性,需进一步验证。本研究对干扰素在抗病毒、提高机体免疫方面的应用提供了理论依据。     

新型猫源干扰素FeIFN-w与干扰素FeIFN-a的表达及抗病毒活性比较

w型干扰素(IFN-w)与a型干扰素(IFN-a)同属于Ⅰ型干扰素, 都具有抗病毒, 抗增殖和免疫调节的功能, 但它们之间的活性却存在较大差异。通过PCR扩增猫w型干扰素基因(feIFN-w), 根据GenBank公布的猫a型干扰素基因序列,合成猫a型干扰素基因(feIFN-a)。分别构建原核表达载体pET-His/feIFN-a和pET-His/feIFN-w, 转化大肠杆菌Rosetta(DE3)进行表达。表达产物经Ni-NTA 亲和层析纯化, 复性后蛋白用细胞病变抑制法进行抗病毒活性测定。结果显示, 重组猫w型干扰素(feIFN-w)抗病毒活性明显高于重组猫a型干扰素(feIFN-a), 尤其对H9N2亚型禽流感病毒(AIV), feIFN-w的活性是feIFN-a的160倍, 对犬瘟热病毒(CDV), feIFN-w的活性是feIFN-a的4倍, 而日本同类产品Intercat?对CDV和AIV均未表现活性。以上研究为以w型干扰素为基础的抗病毒药物应用奠定了重要的理论基础。     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes

Virus infection induces a rapid cellular response in cells characterized by the induction of interferon. While interferon itself does not induce an antiviral response, it activates a number of interferon-stimulated genes that collectively function to inhibit virus replication and spread. Previously, we and others reported that herpes simplex virus type 1 (HSV-1) induces an interferon -independent antiviral response in the absence of virus replication. Here, we report that the HSV-1 proteins ICP0 and vhs function in concert to disable the host antiviral response. In particular, we show that ICP0 blocks interferon regulatory factor IRF3- and IRF7-mediated activation of interferon-stimulated genes and that the RING finger domain of ICP0 is essential for this activity. Furthermore, we demonstrate that HSV-1 modifies the IRF3 pathway in a manner different from that of the small RNA viruses most commonly studied.     

Pterostilbene effectively inhibits influenza A virus infection by promoting the type I interferon production

With the prevalence of novel strains and drug-resistant influenza viruses, there is an urgent need to develop effective and low-toxicity anti-influenza therapeutics. Regulation of the type I interferon antiviral response is considered an attractive therapeutic strategy for viral infection. Pterostilbene, a 3,5-dimethoxy analog of resveratrol, is known for its remarkable pharmacological activity. Here, we found that pterostilbene effectively inhibited influenza A virus infection and mainly affected the late stages of viral replication. A mechanistic study showed that the antiviral activity of pterostilbene might promote the induction of antiviral type I interferon and expression of its downstream interferon-stimulated genes during viral infection. The same effect of pterostilbene was also observed in the condition of polyinosinic-polycytidylic acid (poly I:C) transfection. Further study showed that pterostilbene interacted with influenza non-structural 1 (NS1) protein, inhibited ubiquitination mediated degradation of RIG-I and activated the downstream antiviral pathway, orchestrating an antiviral state against influenza virus in the cell. Taken together, pterostilbene could be a promising anti-influenza agent for future antiviral drug exploitation and compounds with similar structures may provide new options for the development of novel inhibitors against influenza A virus (IAV).     

The nitric oxide pathway provides innate antiviral protection in conjunction with the type I interferon pathway in fibroblasts

The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.     

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.     

Subversion of innate host antiviral strategies by the hepatitis C virus

Since its discovery in 1989, Hepatitis C Virus (HCV) has been recognized as a major cause of chronic hepatitis, end-stage cirrhosis and hepatocellular carcinoma affecting world wide more than 210 million people. The fact that 80% of newly infected patients fail to control infection, the slow development of overt disease and immune-response as well as the unsatisfying results of current IFN/ribavirin combination therapy suggests that the hepatitis C virus developed powerful strategies to evade and to antagonize the immune response of the host and to resist the antiviral actions of interferons. During the last 10 years several viral strategies have been uncovered for control and evasion from cellular antiviral host response initiated by the pathogen-associated molecular pattern recognizing receptors RIG1 and TLR3 and mediated by the release of type I interferon and subsequent induction of interferon stimulated genes. This review highlights recent results providing an idea of how the hepatitis C virus interferes with the different steps of initial antiviral host-response and establishes persistent infection.     

Zika virus inhibits type‐I interferon production and downstream signaling

Zika virus is an emerging mosquito‐borne pathogen that is associated with Guillain–Barré syndrome in adults and microcephaly and other neurological defects in newborns. Despite being declared an international emergency by the World Health Organization, comparatively little is known about its biology. Here, we investigate the strategies employed by the virus to suppress the host antiviral response. We observe that once established, Zika virus infection is impervious to interferon treatment suggesting that the virus deploys effective countermeasures to host cell defences. This is confirmed by experiments showing that Zika virus infection impairs the induction of type‐I interferon as well as downstream interferon‐stimulated genes. Multiple viral proteins affect these processes. Virus‐mediated degradation of STAT2 acts to reduce type‐I and type‐III interferon‐mediated signaling. Further, the NS5 of Zika virus binds to STAT2, and its expression is correlated with STAT2 degradation by the proteasome. Together, our findings provide key insights into how Zika virus blocks cellular defense systems. This in turn is important for understanding pathogenesis and may aid in designing antiviral therapies.     

Novel paradigms of innate immune sensing of viral infections

According to the existing paradigm, cellular recognition of viral infection is mediated by molecular patterns within the virus particle or produced during virus replication. However, there are various physical cellular changes indicative of infection that could also trigger innate antiviral responses. The type-I interferon response is rapidly engaged to limit viral infection and a number of studies have shown that the interferon response, or components of it, are induced by general perturbations to cellular processes. Virus entry requires membrane and cytoskeletal perturbation, and both membrane fusion or actin depolymerising agents alone are able to activate antiviral genes. Viruses cause cellular stress and change the cellular environment, and oxidative stress or endoplasmic reticulum stress will amplify antiviral signaling. Many of these responses converge on interferon regulatory factor 3, suggesting that it plays a crucial role in determining the degree to which the cell responds. This review highlights novel paradigms of viral recognition and speculates that viral infection is sensed as a danger signal.