线粒体抗病毒信号蛋白(MAVS)在宿主天然免疫信号通路中的调节作用

线粒体抗病毒信号蛋白(MAVS)作为一种接头蛋白在调节宿主天然免疫信号通路过程中扮演重要角色.Toll样受体(TLR)和RIG-Ⅰ样受体(RLR)等细胞模式识别受体识别入侵的病原体并将信号传递给MAVS,MAVS通过刺激下游的TBK1复合体和IKK复合体分别活化NF-κB和IRF3等信号通路,进而激活干扰素α/β表达,诱发细胞内抗感染天然免疫反应.MAVS除定位线粒体外,也可定位于过氧化物酶体上.MAVS在细胞内的不同定位决定了其在早期快速和持续性抗病毒天然免疫中的不同调节机制.MAVS只有同时定位在过氧化物酶体和线粒体上才可诱导干扰素刺激基因(ISG)快速且稳定地表达.本文通过对MAVS的发现、结构、细胞定位及其在天然免疫信号通路中的调控机制等最新进展进行综述,以期揭示MAVS蛋白在细胞内天然免疫信号通路中的重要调节作用,为研究病毒逃逸宿主天然免疫的机制和研究新型抗病毒免疫治疗策略提供新思路.

三黄鸡MAVS基因克隆及生物信息学分析

【目的】掌握三黄鸡线粒体抗病毒信号蛋白(MAVS)基因的生物信息学,为MAVS基因诱导表达及禽类固有免疫相关机制研究打下基础。【方法】根据Gen Bank中已公布的原鸡MAVS基因序列设计1对特异性引物,采用RT-PCR扩增三黄鸡MAVS基因,并应用相关生物软件进行序列分析及其蛋白结构预测。【结果】三黄鸡MAVS基因编码区(CDS)全长1926 bp,编码641个氨基酸,与Gen Bank中已发表的原鸡MAVS氨基酸序列(NP_001012911.1)比对,三黄鸡MAVS氨基酸序列在第440、488、506和606位存在4个氨基酸差异,分别为440A→T、488P→L、506I→L和606E→K。三黄鸡MAVS基因与原鸡的相似性最高,达99.0%;与日本鹌鹑的相似性次之,为91.0%;与斑马鱼的相似性最低,仅37.6%。三黄鸡MAVS蛋白分子质量为67.79 ku,理论等电点(p I)为5.04,属于跨膜蛋白,无信号肽序列;其二级结构中折叠占0.5%,无规则卷曲占88.7%,螺旋占10.8%。【结论】三黄鸡MAVS基因序列表现出高度的保守性,可作为今后研究三黄鸡抗病毒机制的重要指标之一。

鸡MAVS(IPS-1)基因的克隆及其对IFN-β激活作用

为研究鸡线粒体抗病毒信号蛋白(mitochondrial anti-viral signaling protein,MAVS)在I型干扰素信号通路中的作用,对克隆的鸡MAVS基因进行了生物信息学分析。结果表明,多数在哺乳动物MAVS中发挥重要作用的蛋白基序及氨基酸位点在鸡中不保守;对鸡MAVS与其他物种进行了进化分析,发现该基因具有较强的种间特异性;DF-1细胞在感染新城疫病毒后12h,MAVS和IFN-β的mRNA水平均被上调,提示鸡MAVS可能参与了细胞抗病毒免疫反应;同时在DF-1细胞中过表达鸡MAVS能够激活IFN-β启动子活性。上述研究揭示了鸡MAVS在诱导I型干扰素产生中具有重要作用,为进一步研究禽类先天性免疫抗病毒途径奠定基础。

p21对MAVS通路的调节

目的:研究MAV S与p21的相互作用,对MAVS通路的调节。方法:我们首先通过PCR的方法克隆了全长的p21基因,然后通过co-IP方法进一步验证p21与MAVS是否可以发生相互作用,接下来通过荧光素酶报道基因的方法研究p21对MAVS通路的影响。结果:通过co-IP发现p21与MAVS可以发生相互作用,另外p21降低MAVS活性。结论:成功地克隆了p21基因,p21与MAVS在293T细胞内可以发生相互作用,p21可以抑制MAVS激活NF-κB途径和IFN-β途径的活性,为进一步研究p21在MAVS通路中所起的作用奠定基础。

猪MAVS基因原核表达载体的构建及其多克隆抗体的制备

通过RT-PCR从PK-15细胞系中扩增克隆MAVS(mitochondrial antiviral signaling protein)基因,构建原核表达载体p ET-MAVS220,转化感受态细胞Rosetta(DE3),利用IPTG诱导表达,重组MAVS经纯化后免疫4周龄昆明系小鼠制备抗线粒体抗病病毒信号蛋白(MAVS)多克隆抗体。诱导表达的最佳条件为IPTG 0.05 mmol/L,37℃诱导6 h,重组MAVS以可溶性蛋白和包涵体两种形式表达。应用该重组蛋白免疫小鼠获得的抗MAVS多克隆抗体与纯化的重组MAVS蛋白反应效价可达1∶16 000;该抗体与Poly(I∶C)刺激PK-15细胞产生的MAVS及与转染了重组MAVS基因真核表达载体pcDNA3.0-MAVS的BHK-21细胞表达的MAVS蛋白发生特异性反应,效价可达1∶1 000,特异性良好。

绵羊MAVS基因的原核表达及多克隆抗体的制备

通过RT-PCR方法从绵羊肺细胞(sheep lung cells,SLT)扩增获得MAVS(mitochondrial antiviral signaling protein)基因,并将其克隆至原核表达载体pET-32a,测序鉴定结果表明成功获得重组质粒pET-32a-MAVS。将其转化至感受肽细胞BL21中,经IPTG诱导获得重组蛋白。将纯化的MAVS蛋白免疫BALB/c雌鼠,制备抗MAVS的鼠源多克隆抗体。SDS-PAGE结果表明该抗体具有良好的反应原性。MAVS多克隆抗体的成功制备为其生物学功能研究奠定了基础。

Suppression of Innate Immune Signaling Molecule,MAVS,Reduces Radiation-induced Bystander Effect

Although radiation induced bystander effect(RIBE)has been investigated for decades for secondary cancer risk assessment during cancer radiotherapy,the underlying gene regulation remains unclear,especially the roles of immune factors in RIBE.Mitochondrial antiviral signaling(MAVS)protein.

抑制免疫分子MAVS缓解辐射引起的线粒体功能紊乱

Mitochondrial antiviral signaling(MAVS)protein is signaling adaptor with antiviral feature and locate in the mitochondrial out-membrane.Our study demonstrated that knockdown of MAVS increases the radioresistance and irradiation(IR)induced the change of MAVS expression in cells.Knockdown of MAVS alone could decrease the mitochondrial membrane potential,while increase the mitochondrial ATP production and the expressions of apoptosis related proteins.While knockdown of MAVS followed by X-rays increased the cellular mitochondrial membrane potential,ATP production and expression of apoptosis protein,compared to the IR group only.

MAVS在神经内分泌肿瘤诊断和预后临床价值的探讨

目的神经内分泌肿瘤是威胁患者生命的神经系统疾病。神经内分泌肿瘤患者免疫力下降是造成肿瘤恶化的原因之一。神经内分泌肿瘤与免疫分子MAVS的关系尚需研究。方法以我院肿瘤科收治的468名神经内分泌肿瘤患者为研究对象(包括神经内分泌肿瘤患者207例和重症神经内分泌肿瘤患者161例),同时以207例健康志愿者作为对照。使用western blot检测治疗前后神经内分泌肿瘤患者和健康志愿者血细胞中MAVS的水平。分析MAVS的水平与神经内分泌肿瘤之间的相关性。结果神经内分泌肿瘤患者血细胞中MAVS的蛋白水平显著高于健康志愿者血细胞中MAVS的蛋白水平(P<0.05)。治疗后神经内分泌肿瘤患者血细胞中MAVS的蛋白表达水平下降。重度神经内分泌肿瘤患者血细胞中MAVS的蛋白水平显著高于一般神经内分泌肿瘤患者血细胞中MAVS的蛋白水平(P<0.05)。神经内分泌肿瘤患者血细胞中MAVS的蛋白水平与神经内分泌肿瘤的恶化程度具有正相关性。结论神经内分泌肿瘤患者血细胞中MAVS可能是神经内分泌肿瘤患者的特异生物标志物。MAVS水平与神经内分泌肿瘤的恶化程度具有密切正相关性。

MAVS-loaded unanchored Lys63-linked polyubiquitin chains activate the RIG-I-MAVS signaling cascade

The adaptor molecule MAVS forms prion-like aggregates to govern the RIG-I-like receptor(RLR)signaling cascade.Lys63(K63)-linked polyubiquitination is critical for MAVS aggregation,yet the underlying mechanism and the corresponding E3 ligases and deubiquitinating enzymes(DUBs)remain elusive.Here,we found that the K63-linked polyubiquitin chains loaded on MAVS can be directly recognized by RIG-I to initiate RIG-I-mediated MAVS aggregation with the prerequisite of the CARDRIG-I-CARDMAVS interaction.Interestingly,many K63-linked polyubiquitin chains attach to MAVS via an unanchored linkage.We identified Ube2N as a major ubiquitin-conjugating enzyme for MAVS and revealed that Ube2N cooperates with the E3 ligase Riplet and TRIM31 to promote the unanchored K63-linked polyubiquitination of MAVS.In addition,we identified USP10 as a direct DUB that removes unanchored K63-linked polyubiquitin chains from MAVS.Consistently,USP10 attenuates RIG-I-mediated MAVS aggregation and the production of type I interferon.Mice with a deficiency in USP10 show more potent resistance to RNA virus infection.Our work proposes a previously unknown mechanism for the activation of the RLR signaling cascade triggered by MAVS-attached unanchored K63-linked polyubiquitin chains and establishes the DUB USP10 and the E2:E3 pair Ube2N-Riplet/TRIM31 as a specific regulatory system for the unanchored K63-linked ubiquitination and aggregation of MAVS upon viral infection.

Large yellow croaker(Larimichthys crocea)mitofusin 2 inhibits type I IFN responses by degrading MAVS via enhanced K48‑linked ubiquitination

In mammals,mitofusin 2(MFN2)is involved in mitochondrial fusion,and suppresses the virus-induced RIG-I-like receptor(RLR)signaling pathway.However,little is known about the function of MFN2 in non-mammalian species.In the present study,we cloned an MFN2 ortholog(LcMFN2)in large yellow croaker(Larimichthys crocea).Phylogenetic analysis showed that MFN2 emerged after the divergence of amphioxus and vertebrates.The protein sequences of MFN2 were well conserved from fsh to mammals.LcMFN2 was expressed in all the tissues/organs examined at diferent levels,and its expression was upregulated in response to poly(I:C)stimulation.Overexpression of LcMFN2 inhibited MAVS-induced type I interferon(IFN)promoter activation and antiviral gene expression.In contrast,knockdown of endogenous LcMFN2 enhanced poly(I:C)induced production of type I IFNs.Additionally,LcMFN2 enhanced K48-linked polyubiquitination of MAVS,promoting its degradation.Also,overexpression of LcMFN2 impaired the cellular antiviral response,as evidenced by the increased expression of viral genes and more severe cytopathic efects(CPE)in cells infected with spring viremia of carp virus(SVCV).These results indicated that LcMFN2 inhibited type I IFN response by degrading MAVS,suggesting its negative regulatory role in cellular antiviral response.Therefore,our study sheds a new light on the regulatory mechanisms of the cellular antiviral response in teleosts.

MAVS^(-/-)细胞系的构建及其影响禽流感病毒复制的研究

本研究利用CRISPR/Cas9基因编辑技术构建MAVS基因稳定敲除的细胞株,对流感病毒的增殖特性进行初步研究。设计、构建靶向MAVS基因sgRNA表达载体,与pCAG-Cas9-EGFP表达载体共转染MDCK细胞,经过流式细胞仪分选、PCR、基因测序筛选MAVS敲除细胞系,CCK-8法检测细胞增殖速度;荧光定量PCR方法检测H9N2亚型禽流感病毒(AIV)感染后的TCID_(50)、病毒拷贝数以及IRF3、IFN-β、Mx1基因转录水平变化。结果显示,筛选出1株MAVS基因缺失44 bp的MDCK细胞(MAVS^(-/-)MDCK),其增殖速度与正常细胞相比未观察到显著差异;荧光定量PCR结果表明,TCID_(50)、病毒拷贝数差异最高可分别达到MDCK细胞的4.11倍和1.82倍;MAVS^(-/-)MDCK中IRF3、IFN-β和Mx1 m RNA表达水平显著降低,表明MAVS敲除后抑制了Ⅰ型干扰素信号通路。表明,本研究获得的MAVS^(-/-)MDCK能够促进禽流感病毒的复制,为提高疫苗生产效率和质量提供候选细胞株;该细胞株也为进一步研究MAVS参与抗病毒天然免疫应答奠定基础。

人MAVS小干扰RNA质粒的构建及稳定干扰MAVS细胞株的筛选

目的:构建人线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)的小干扰RNA真核表达质粒,并筛选出稳定干扰MAVS的细胞株。方法:根据文献报道的序列和载体的黏性末端,设计合成2条针对MAVS的DNA序列,退火后连接到载体pSUPER.retro.neo^+gfp上,经测序分析正确后,质粒命名为psiMAVS。用脂质体转染质粒到MCF-7细胞中,经G418加压筛选稳定表达MAVS小干扰RNA的细胞株,用免疫印迹检测细胞中MAVS的表达情况,将干扰效果好的细胞株命名为MCF-7/siMAVS,再用荧光素酶试验检测MCF-7/siMAVS细胞对外源MAVS或poly(dA-dT)诱导干扰素-β(IFN-β)转录的情况。结果:免疫印迹结果证实建立的稳定细胞株MCF- 7/siMAVS能够有效干扰MAVS的表达,并在外源MAVS或poly(dA-dT)存在的情况下,抑制IFN-β的转录活性。结论:构建了表达MAVS小干扰RNA的质粒psiMAVS。筛选出稳定干扰MAVS表达的细胞株MCF-7/siMAVS,为深入研究MAVS在先天免疫中的作用提供了平台。

SARS-CoV-2 membrane glycoprotein M antagonizes the MAVS-mediated innate antiviral response

A novel SARS-related coronavirus(SARS-CoV-2)has recently emerged as a serious pathogen that causes high morbidity and substantial mortality.However,the mechanisms by which SARS-CoV-2 evades host immunity remain poorly understood.Here,we identified SARS-CoV-2 membrane glycoprotein M as a negative regulator of the innate immune response.We found that the M protein interacted with the central adaptor protein MAVS in the innate immune response pathways.This interaction impaired MAVS aggregation and its recruitment of downstream TRAF3,TBK1,and IRF3,leading to attenuation of the innate antiviral response.Our findings reveal a mechanism by which SARS-CoV-2 evades the innate immune response and suggest that the M protein of SARSCoV-2 is a potential target for the development of SARS-CoV-2 interventions.

猪线粒体抗病毒信号蛋白MAVS对口蹄疫病毒感染宿主的影响

采用构建猪源MAVS重组质粒pEGFP-MAVS,将其转染PK-15细胞24h后,再感染口蹄疫病毒(MOI=0.1),通过Western blot和real-time PCR检测MAVS融合蛋白表达及对口蹄疫病毒感染宿主细胞的复制和病毒感染滴度。结果显示:重组质粒pEGFP-MAVS能在PK-15细胞中获得表达,并且上调MAVS基因对口蹄疫病毒在宿主细胞中的早期复制有显著抑制作用,病毒的感染滴度也有一定的降低。结果表明:MAVS具有一定的抗病毒作用,此为进一步研究口蹄疫病毒逃逸宿主天然免疫的机制奠定了基础。

线粒体抗病毒信号蛋白MAVS真核表达质粒的构建及表达

目的:构建线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)真核表达质粒。方法:从人胚肾细胞系HEK293T细胞的总RNA中经过逆转录和PCR获得MAVS的全长cDNA双链片段,经过酶切后连接到真核表达载体pcDNA3/flag中,挑选出转化生成的阳性克隆进行测序,将序列正确的重组质粒命名为pcDNA3/flag-MAVS。借助脂质体转染pcDNA3/flag-MAVS质粒到HEK293T细胞中,用Western印迹检测目的蛋白的表达。同时用β干扰素的荧光素酶报告基因(IFN-β-luc)检测MAVS蛋白活性。结果:Western印迹实验证明pcDNA3/flnag-MAVS可以在HEK293T细胞中表达MAVS,并且能使IFN-β报告基因活性明显增强。结论:构建了重组质粒pcDNA3/flag-MAVS,在细胞中表达MAVS后具有刺激IFN-β转录的生物活性。

SARS-CoV-2 ORF10 suppresses the antiviral innate immune response by degrading MAVS through mitophagy

The global coronavirus disease 2019(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused severe morbidity and mortality in humans.It is urgent to understand the function of viral genes.However,the function of open reading frame 10(ORF10),which is uniquely expressed by SARS-CoV-2,remains unclear.In this study,we showed that overexpression of ORF10 markedly suppressed the expression of type I interferon(IFN-I)genes and IFN-stimulated genes.Then,mitochondrial antiviral signaling protein(MAVS)was identified as the target via which ORF10 suppresses the IFN-I signaling pathway,and MAVS was found to be degraded through the ORF10-induced autophagy pathway.Furthermore,overexpression of ORF10 promoted the accumulation of LC3 in mitochondria and induced mitophagy.Mechanistically,ORF10 was translocated to mitochondria by interacting with the mitophagy receptor Nip3-like protein X(NIX)and induced mitophagy through its interaction with both NIX and LC3B.Moreover,knockdown of NIX expression blocked mitophagy activation,MAVS degradation,and IFN-I signaling pathway inhibition by ORF10.Consistent with our observations,in the context of SARS-CoV-2 infection,ORF10 inhibited MAVS expression and facilitated viral replication.In brief,our results reveal a novel mechanism by which SARS-CoV-2 inhibits the innate immune response;that is,ORF10 induces mitophagy-mediated MAVS degradation by binding to NIX.

Novel insights into stress-induced susceptibility to influenza:corticosterone impacts interferon-βresponses by Mfn2-mediated ubiquitin degradation of MAVS

Although stress has been known to increase the susceptibility of pathogen infection,the underlying mechanism remains elusive.In this study,we reported that restraint stress dramatically enhanced the morbidity and mortality of mice infected with the influenza virus(H1N1)and obviously aggravated lung inflammation.Corticosterone(CORT),a main type of glucocorticoids in rodents,was secreted in the plasma of stressed mice.We further found that this stress hormone significantly boosted virus replication by restricting mitochondrial antiviral signaling(MAVS)protein-transduced IFN-βproduction without affecting its mRNA level,while the deficiency of MAVS abrogated stress/CORT-induced viral susceptibility in mice.Mechanistically,the effect of CORT was mediated by proteasome-dependent degradation of MAVS,thereby resulting in the impediment of MAVS-transduced IFN-βgeneration in vivo and in vitro.Furthermore,RNA-seq assay results indicated the involvement of Mitofusin 2(Mfn2)in this process.Gain-and loss-offunction experiments indicated that Mfn2 interacted with MAVS and recruited E3 ligase SYVN1 to promote the polyubiquitination of MAVS.Co-immunoprecipitation experiments clarified an interaction between any two regions of Mfn2(HR1),MAVS(C-terminal/TM)and SYVN1(TM).Collectively,our findings define the Mfn2-SYVN1 axis as a new signaling cascade for proteasome-dependent degradation of MAVS and a‘fine tuning’of antiviral innate immunity in response to influenza infection under stress.

MicroRNA-33/33^(*) inhibit the activation of MAVS through AMPK in antiviral innate immunity

Innate immunity plays a prominent role in the host defense against pathogens and must be precisely regulated.As vital orchestrators in cholesterol homeostasis,microRNA-33/33*have been widely investigated in cellular metabolism.However,their role in antiviral innate immunity is largely unknown.Here,we report that VSV stimulation decreased the expression of miR-33/33*through an IFNAR-dependent manner in macrophages.Overexpression of miR-33/33*resulted in impaired RIG-I signaling,enhancing viral load and lethality whereas attenuating type I interferon production both in vitro and in vivo.In addition,miR-33/33*specifically prevented the mitochondrial adaptor mitochondrial antiviral-signaling protein(MAVS)from forming activated aggregates by targeting adenosine monophosphate activated protein kinase(AMPK),subsequently impeding the mitophagy-mediated elimination of damaged mitochondria and disturbing mitochondrial homeostasis which is indispensable for efficient MAVS activation.Our findings establish miR-33/33*as negative modulators of the RNA virus-triggered innate immune response and identify a previously unknown regulatory mechanism linking mitochondrial homeostasis with antiviral signaling pathways.

Attitude Determination for MAVs Using a Kalman Filter

This paper presents a Kalman filter to effectively and economically determine the Euler angles for micro aerial vehicles （MAVs）, whose size and payload are severely limited. The filter uses data from a series of micro-electro mechanical system sensors to determine the selected 3 variables of the direction cosine matrix and the bias of the rate gyro sensors as state elements in a dynamic model, with the gravitational acceleration to build a measurement model. For high speed maneuvers, rigid motion equations are used to correct the measurements of the gravitational acceleration. The filter is designed to automatically tune its gain based on the dynamic system state. Simulations indicate that the Euler angles can be determined with standard deviations less than 3°. The algorithm was successfully implemented in a miniature attitude measurement system suitable for MAVs. Aerobatic flights show that the attitude determination algorithm works effectively. The attitude determination algorithm is effective and economical, and can also be applied to bionic robofishs and land vehicles, whose size and payload are also greatly limited.