摘要
冠状病毒是人和动物的重要致病原,其中新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)给人类健康带来了致命威胁。宿主固有免疫反应是宿主抵抗病原体入侵的第一道防线,但过激的免疫应答也会加重病毒感染和病理损伤。病毒免疫逃逸是冠状病毒的重要致病机制。本文主要从宿主免疫传感器、干扰素、细胞因子和冠状病毒免疫逃逸方面重点阐述了冠状病毒的致病机制,以期为抗冠状病毒药物的研发提供理论参考。
冠状病毒(coronavirus, CoV)是具有囊膜的单股正链RNA病毒,囊膜表面覆有12~24 nm的棒状突起,形如花冠,故称冠状病毒。冠状病毒属于尼多病毒目冠状病毒科,根据基因组结构相似性及血清型不同,主要分为α、β、γ和δ 4个属。其中,α属主要包括人冠状病毒(human coronavirus,HCoV-229E和HCoV- NL63)、猪传染性腹泻病毒(porcine epidemic virus, PEDV)及犬冠状病毒(canine coronavirus, CCoV)等。β属主要包括2019年暴发的新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)、严重急性呼吸系统综合征冠状病毒(severe acute respiratory syndrome coronavirus, SARS-CoV)、中东呼吸综合征(middle east respiratory syndrome, MERS)及小鼠肝炎病毒(murine hepatitis virus, MHV)等。γ属主要包括传染性支气管炎病毒(infection bronchitis virus, IBV)。δ属主要包括猪δ冠状病毒(porcine deltacoronavirus, PDCoV
现已研究表明,与其他已知病毒相似,冠状病毒侵入宿主也主要通过宿主细胞表面的膜受体介导。随着近年来基因敲除技术与分子靶点技术的进步,多种介导冠状病毒入侵宿主的蛋白受体被发现,其中α属HCoV-229E和δ属PDCoV等冠状病毒主要通过宿主细胞表面的氨基肽酶N (aminopeptidase N, APN)受体介导入侵细胞,而SARS-CoV-2和SARS-CoV等β属冠状病毒主要通过其囊膜表面的S刺突蛋白与宿主细胞膜表面的ACE2受体相结合而侵入宿主纤毛支气管上皮细胞和Ⅱ型肺泡细
冠状病毒为直径为60~220 nm的正链RNA病毒,其RNA长度为27~32 kb,5′端带有甲基化帽状结构,3′端具有多聚腺苷核糖核酸尾(PolyA),主要分为复制酶编码区和结构蛋白编码区。在受体介导下冠状病毒通过膜融合途径将携带的病毒RNA释放入宿主细胞中,首先通过复制酶编码区翻译合成RNA依赖的RNA聚合酶,随即在RNA聚合酶作用下,以病毒基因组为模板合成负链RNA,然后再通过负链RNA合成新的病毒基因组和亚基因组,起始子代病毒和病毒基因组的合成。冠状病毒的5′端2/3部分的复制酶编码区含有2个关键开放阅读框(open reading frames, ORFs),ORF1a和ORF1b,它们可通过不同的剪切编码16个保守的非结构蛋白(nsp1-nsp16),其基因组后1/3部分主要编码4个结构蛋白:刺突蛋白(spike protein, S)、包膜蛋白 (envelope protein, E)、膜糖蛋白(membrane glycoprotein, M)和核衣壳蛋白(nucleocapsid protein, N
冠状病毒入侵宿主引发过激的免疫反应是其临床高致死性的主要致病机制。现有的研究表明冠状病毒感染宿主后释放的病毒基因组及相关的病毒蛋白会被宿主细胞中的模式识别受体(pattern recognition receptor, PRR)所识别,而宿主细胞感知病原微生物入侵后会通过调控体内免疫信号通路诱导如Ⅰ型干扰素(interferon I, IFN-I)、促炎因子及多种抗病毒基因的表达而抑制病毒复制。然而,为对付宿主这种先天性抗病毒免疫反应,多数种类的冠状病毒在感染宿主的长期进化过程中逐渐形成了一套复杂的免疫逃逸机制,或通过自身编码的蛋白拮抗或延迟宿主抗病毒干扰素系统的表
天然免疫反应是机体在长期进化过程中形成的抵抗外源微生物入侵的第一道屏障。作为宿主固有免疫体系不可或缺的模式识别受体PRR在机体抵抗病原微生物感染过程中扮演关键作用。机体受到病原微生物感染后,细胞中的PRR能特异性识别病原微生物的携带的如细菌脂多糖、肽聚糖、脂蛋白、鞭毛蛋白、病毒双链RNA、非甲基化CpG DNA等病原相关分子模式(pathogen-associated molecular patterns, PAMPs),进而诱导先天性免疫信号级联反应,继而启动宿主的先天性免疫反
TLRs是一种跨膜蛋白模式识别受体,其胞内区含有TLRs和白细胞介素1(IL-1)受体家族特有的结构域TIR,其包外区含有富含亮氨酸重复序列,这是决定其特异性的关键结构域。TLRs在细胞中的位置决定了它们识别不同PMAP的功能,内体中的TLRs主要负责识别核酸,如TLR7/8主要识别单链RNA (ssRNA)、TLR9主要识别CpG DNA,而质膜上的TLRs (TLR1和TLR2)主要负责识别病毒的蛋白和脂质等成
RLRs是一种细胞质传感器,包括RIG-I、黑素瘤分化相关基因5 (melanoma differentiation factors 5, MDA5)和LGP2(laboratory of genetics and physiology 2)3种类型,此类传感器可特异性识别细胞中的多种病毒dsRNA。RIG-I含有2个N端招募激活结构域(caspase activation and recruitment domain, CARDs)、1个中间解旋酶核心和1个C端结构域(c-terminal domain, CTD),其中间解旋酶核心区含有解旋酶结构1 (helicase 1,Hel-1)、解旋酶结构2 (helicase 2, Hel-2) 和中间插入区(helicase 2i, Hel-2i
先天性免疫反应是机体抗冠状病毒感染的第一道防线。IFN-I是宿主抗病毒的先天性免疫调节因子,当细胞受到病毒感染时,IFN主要通过与效应细胞表面的膜受体(IFNAR1和IFNAR2)结合,进而激活酪氨酸激酶与活化转录因子(janus kinases (JAKs)-signal transducer and activator of transcription(STAT),JAK-STAT)信号转导通路而发挥抗病毒活性。STAT由STAT1、STAT2和STAT3 3种亚型构成,受刺激活化的STAT1与STAT2可形成同源二聚体,募集IFN调节因子9 (interferon regulation factor 9,RF9),最终促进IFN刺激因子3(interferon stimulated genes factor 3,ISGF-3)的形成,活化的ISGF-3入核后与ISGs启动子中的IFN刺激的应答元件(interferon-stimulated response element,ISRE)结合,促进ISGs的表达。现已研究表明ISGs具有广谱的抗病毒活性和调节适应性免疫作用,在机体抵抗病毒入侵过程中扮演关键作用。例如,2',5'-寡聚腺苷酸合成酶(2′,5′-oligoadenylate synthetase,OAS)和蛋白激酶(protein kinase,PKR)是最早发现的dsRNA依赖性激酶,其中OAS主要通过降解病毒和宿主的RNA,抑制蛋白合成,进而发挥抗病毒活性,而PKR主要通过磷酸化激活真核细胞翻译起始因子eIF2α (eukaryotic initiation factor 2α),发挥抗病毒作用;人类MxA蛋白在进入细胞后立即与病毒核衣壳靶向结合,降低病毒核酸酶的活性,在感染早期干扰病毒复制;其他抗病毒ISGs产物,如干扰素诱导跨膜蛋白(IFITM proteins)、25-羟基氧化酶(CH25H)、锌指抗病毒蛋白(ZAP)等也与冠状病毒感染高度相
冠状病毒感染可诱发宿主产生过激细胞因子风暴(cytokine storm),导致严重的急性肺损伤、急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS),甚至多器官功能衰竭和死亡。临床研究证实患者血清中细胞因子和趋化因子的表达水平与疾病预后密切相关。研究发现新冠病毒SARS-CoV-2 感染患者血浆中存在白细胞介素-6 (IL-6)、白细胞介素-7 (IL-7)、白细胞介素-10 (IL-10)、粒细胞集落刺激因子(granulocyte colony stimulating factor, G-SCF)、趋化因子CXCL-10 (interferon-inducible protein-10, IP-10)、单核细胞趋化蛋白(membrane cofactorprotein-1, MCP-1)、肿瘤坏死因子-α(tumor necrosis factor, TNF-α)及巨噬细胞炎症蛋白(macrophage inflammatory protein-1 alpha, MIP-1a)异常升
在与宿主的长期斗争中,冠状病毒已进化多种应对天然免疫系统的机制,主要包括逃避和拮抗两种策略(

Figure1 Potential mechanism of coronavirus against innate immune response
逃避宿主的天然免疫识别是外源微生物感染宿主的有效策略。冠状病毒复制时产生的大量中间体dsRNA会激活PRR,使临近细胞启动先天性免疫反应。冠状病毒为了逃避先天性免疫反应必须躲过PRR的识别。研究发现,SARS-CoV、MHV等多种冠状病毒可在一种具双层膜的小囊泡中进行复制,其有可能是为了掩藏了病毒PAMP,避免被细胞质中PRR所识别。N7-鸟苷甲基化的帽子(cap,包括cap-0和cap-1)是真核生物mRNA的结构特征和RNA聚合酶Ⅱ转录产物的标志,宿主可用来识别自我与非我RNA。为逃避宿主PRR识别,许多病毒模仿宿主加帽机制对自身RNA进行修饰。体外实验表明,SARS-CoV的非结构蛋白nsp14和nsp16/nsp10复合物参与其RNA加帽过
拮抗宿主的先天性免疫反应也是外源微生物抵抗宿主免疫反应的有效策略。研究发现,冠状病毒蛋白可通过多种机制直接或间接抑制干扰素及其介导的免疫信号通路。临床研究表明冠状病毒感染患者的IFN-I水平普遍偏低,感染SARS-CoV-2、SARS-CoV和MERS-CoV的重症患者更
冠状病毒编码的非结构蛋白nsps能显著抑制IFN-I产生及其信号传导通路,遏制宿主的先天性免疫反应。例如SARS-CoV的nsp1可选择性诱导细胞内核酸内切酶剪切宿主mRNA的5'-UTR,使宿主mRNA失去转录活性,而与核糖体40S亚基结合,可抑制核糖体40S亚基与60S亚基的结合,进而抑制宿主mRNA的翻译功
冠状病毒的结构蛋白不仅是病毒结构必不可少的组分,而且在拮抗宿主的干扰素传导途径中发挥重要作用。最新研究发现,SARS-CoV-2编码的M蛋白可以与RIG-I、MAVS和TBK1相互作用,阻止RIG-I、MAVS、TRAF3和TBK1蛋白复合体的形成,进而抑制IRF3的磷酸化、入核,以及I型和III干扰素诱导的转录激
冠状病毒编码的辅助蛋白具有广泛调控宿主免疫的功能,与病毒的致病性密切相关。SARS-CoV-2编码的ORF6、ORF8和ORF3b是强效干扰素拮抗剂,在SARS-CoV-2感染早期,可阻碍IFN的释放,干扰宿主抗病毒免疫反应,促进病毒复制。SARS-CoV编码的ORF3a/3b、ORF6、ORF7a/7b、ORF8a/8b和ORF9b等8种辅助蛋白中ORF3b和ORF6业已被证实具有拮抗IFN作用,其中ORF3b可抑制RIG-I、MAVS介导的转录因子IRF3和NF-kB的活化,拮抗IFN产生,而ORF6主要通过阻止STAT1与核转运蛋白β1(KPNB1)结合形成复合物导致细胞质中STAT1无法入核而抑制IFN信号传导通路活
冠状病毒是目前已知的拥有最长基因组的RNA病毒,其不仅在自然界广泛存在,而且是多种哺乳动物和鸟类的呼吸道常见病原。近十多年来,冠状病毒相继在人类引起3次世界范围内的流行,即2003年的SARS-CoV,2012年流行至今的MERS-CoV和2019年暴发至今的SARS-CoV-2,其中SARS-CoV-2的传染性最强,迄今已致全球8 000多万人感染,170多万人死亡,且全球感染仍未得到有效控制。新冠病毒SARS-CoV-2作为首次感染人类的新病原,人类目前对其宿主、传染源及致病机制等仍不十分清楚,迄今尚无防治的有效药物。据报道,SARS-CoV-2与SARS-CoV基因组序列相似率高达79.5%,感染症状也极其相似。机体的免疫失调和病毒免疫逃避是SARS-CoV的主要致病机制,除了抗病毒治疗外,适度的免疫调节可改善重症患者的预后,降低病死率。同样的,SARS-CoV-2患者体内多种细胞因子水平异常,大量免疫细胞和组织液聚集肺部,导致呼吸道阻塞和严重的肺损伤。借鉴SARS-CoV的致病机制和药物研发经验,有可能降低研发成本、缩短研发周期,早日开发出针对SARS-CoV-2的特效药物。
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