人类新发NL63冠状病毒木瓜样蛋白酶调控宿主抗病毒天然免疫反应及其分子机制
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摘要
宿主免疫系统分为天然免疫(Innate immunity)和获得性免疫(Adaptive immunity)。天然免疫是非特异形式的免疫应答,是以识别和清除病原体为主的第一道防御屏障。天然免疫反应主要发生在病毒感染的早期且与抗病毒感染紧密相关。病毒入侵后,宿主会立即启动天然免疫应答,通过细胞表面及细胞质中的模式识别受体(PRR)检测各种病原体相关分子模式(PAMP),例如病毒RNA,细菌细胞壁成分等,启动一系列的细胞信号转导级联反应。活化的模式识别受体通过同型互作与衔接蛋白相接,启动信号转导。信号转导过程中通过磷酸化及泛素化修饰激活信号通路中一些重要的基因,诱导蛋白激酶(如IKK?-IKK?-IKK?和TBK1/IKK?激酶复合体)的活化,从而激活转录因子(如NF-?B, IRF7, IRF3),最终引发促炎症细胞因子及I型干扰素基因的转录,共同介导抗病毒效应。
冠状病毒是一类拥有庞大RNA基因组的包膜病毒。至今发现的人类冠状病毒包括HCoV-229E、HCoV-OC43、SARS-CoV、HCoV-NL63和HcoV-HKU1五种,其中NL63和HKU1是继SARS冠状病毒后出现的两种人类新发冠状病毒。NL63冠状病毒是2004年由荷兰学者Van der Hoek L首先报道的一种单股正链RNA病毒,呈世界性分布,流行病学检测发现,NL63冠状病毒在儿童和成人(特别是儿童)呼吸道感染中的阳性检测率高,已成为人类呼吸道感染的主要新发病原之一。
冠状病毒与宿主间的相互作用决定了其致病性和免疫特性。冠状病毒感染宿主后会立即启动抗病毒天然免疫反应,病毒为了躲避宿主机体复杂的防御体系,必然对宿主细胞内的信号转导进行干预和调节。近年来,越来越多的研究表明,病毒通过编码多种功能蛋白调控宿主的抗病毒天然免疫反应。例如,小核糖核酸病毒编码的3C蛋白酶一方面剪切复制聚合酶前体,另一方面抑制RIG-I和NF-?B的活化。我们前期研究表明,NL63冠状病毒nsp3编码的木瓜样蛋白酶(PLP)包括两个核心结构域PLP1和PLP2。通过体内DUB活性分析发现,只有PLP2核心结构域具有DUB活性,而PLP1则没有。PLP2作为一种干扰素拮抗剂,通过去除RIG-I和STING的泛素化修饰,负调节干扰素表达,而且PLP2的DUB活性与其干扰素拮抗作用是相互独立的。由于编码PLP2的nsp3位于细胞内质网(ER)膜上,因此NL63冠状病毒nsp3编码的木瓜样蛋白酶应该是膜定位的。目前对于NL63冠状病毒编码的木瓜样蛋白酶(PLP)调控宿主的抗病毒天然免疫反应机制,以及可溶性木瓜样蛋白酶(PLP2)与膜定位的木瓜样蛋白酶(PLP2-TM)之间的差异性尚不清楚。围绕这一问题我们针对NL63冠状病毒编码的膜定位木瓜样蛋白酶(PLP-TM)进行更加深入的研究,主要结果如下:
一. NL63冠状病毒编码的木瓜样蛋白酶(PLP)在细胞内的定位研究
(一)利用分子克隆及基因定点突变技术,分别构建PLP1-TM, PLP2-TM的重组体及蛋白酶活性催化位点突变体(PLP1-TM C1062A, PLP1-TM H1212A, PLP1-TM D1225A, PLP2-TM C1678A, PLP2-TM H1836A, PLP2-TM D1489A)。
(二)对NL63 PLP蛋白酶进行进行免疫荧光染色,确定了nsp3编码的可溶性PLP和膜定位的PLP-TM在细胞的定位情况。结果表明膜定位前后蛋白表达位置发生改变,PLP均匀的分布于细胞质中,而PLP-TM则集中分布于核周内质网上。同时,我们通过western blot检测到PLP-TM及酶活性突变体的表达条带。
二. PLP-TM的生物学特性研究
(一)利用DUB检测技术对NL63 PLP的去泛素化活性进行分析,发现PLP1-TM和PLP2-TM具有明显的DUB活性,证实PLP具有TM依赖的DUB活性;同时,PLP2-TM的蛋白酶催化活性位点(C, H, D)突变后,其DUB活性降低,其中C1678A和H1836的对野生型PLP2-TM的DUB活性影响较大,而D1849A没有影响。说明PLP2-TM的DUB活性不完全依赖其蛋白酶的催化活性。
(二)利用DUB和去ISG检测技术对NL63 PLP的底物特异性进行研究,发现PLP2-TM对Ub-K48和Ub-K63连接的多聚泛素化没有表现出特异性;其次,以类泛素分子(SUMO和ISG15)为作用底物,发现PLP2和PLP2-TM均具有去ISG活性,然而只有膜定位的PLP(PLP1-TM和PLP2-TM)具有去SUMO活性,说明膜定位对于NL63冠状病毒编码木瓜样蛋白酶的功能发挥具有重要作用。
三. PLP-TM负调控宿主干扰素表达活性研究
(一)利用双荧光素酶报告基因检测技术对PLP-TM干扰素拮抗活性进行分析,发现PLP1-TM和PLP2-TM可以下调外界条件(仙台病毒,RIG-IN等)刺激下I型干扰素的表达。说明膜定位木瓜样蛋白酶(PLP-TM)是一种干扰素拮抗剂而且PLP2-TM的拮抗活性强于PLP1-TM。
(二)对PLP2-TM干扰拮抗活性的特异性进行研究,证实PLP2-TM负调节干扰素表达的作用具有剂量依赖性,而且PLP2-TM的三种蛋白酶活性突变体(PLP2-TM C1678A, PLP2-TM H1836A, PLP2-TM D1489A)均能抑制干扰素产生,且存在剂量效应。说明PLP2-TM的干扰素拮抗活性是独立于蛋白酶活性之外的一种特异性功能。
四. PLP2-TM负调控宿主抗病毒天然免疫反应分子机制研究
(一)首先,利用干扰素表达通路中重要调节蛋白激活干扰素表达,同时瞬转PLP2-TM后检测PLP2-TM对信号蛋白介导的干扰素表达通路的调节作用,结果显示PLP2-TM及其突变体明显抑制由RIG-I/TRAF3/TBK1/IRF3激活的干扰素的产生;说明PLP2-TM干扰素拮抗活性不依赖其蛋白酶催化活性;其次,通过免疫共沉淀技术分析,发现PLP2-TM通过去除RIG-I/TRAF3/TBK1/IRF3的泛素化或ISG化修饰负调控干扰素表达通路。
(二)首先,利用免疫共沉淀技术,对最新发现的干扰素刺激因子STING上下游信号蛋白之间的相互作用进行检测,发现PLP2-TM能够破坏STING与IPS-1间的相互作用,但是不破坏STING与TRAF3, STING与TBK1, IPS-1与TRAF3间的相互作用。说明PLP2-TM阻断了信号由IPS-1向STING的流动,以及STING向下游蛋白分子的传递;其次,我们对STING与蛋白激酶间的相互作用进行检测,发现PLP2-TM能够破坏STING与IKK?间的相互作用,但是PLP2-TM不能破坏STING与TBK1间以及TBK1与IRF3间的相互作用,说明PLP2-TM特异性的阻止了STING募集蛋白激酶IKK?至IPS-1,抑制IRF3的活化。
(三)首先,利用免疫共沉淀技术对STING的泛素化及ISG化进行检测,发现PLP2-TM能够破坏STING的泛素化修饰和ISG化修饰;其次,二聚化是STING激活干扰素表达的一个必要条件,而且STING的泛素化和ISG化是在其二聚化基础上进行的,所用利用不同标签的STING进行IP分析,发现PLP2-TM能够破坏STING的二聚化从而抑制IRF3介导的干扰素表达通路。
本课题研究从多方面多角度阐明了PLP2-TM调控宿主抗病毒天然免疫反应机制。实验阐明了NL63编码的木瓜样蛋白酶(PLP)是一种多功能蛋白酶,除了经典的蛋白酶水解活性,还具有TM依赖的去泛素化酶(DUB)活性,同时又作为一种高效的干扰素拮抗剂负调控宿主的抗病毒天然免疫反应。NL63编码PLP2-TM调控宿主抗病毒天然免疫反应机制研究表明,一方面,PLP2-TM通过DUB活性,去除信号通路中重要调节蛋白的泛素化活性,负调控干扰素表达;另一方面,PLP2-TM能够破坏最新发现的干扰素刺激因子STING与IPS-1间, STING与IKK?间的相互作用,阻止信号由STING流向TRAF3和IKK?,从而阻碍了转录因子IRF3的活化,最终抑制I型干扰素的表达。重要的是,我们首次证实PLP2-TM通过破坏STING的二聚化抑制STING的干扰素激活作用,为病毒调控宿主抗病毒天然免疫反应机制研究提供有力参考资料。综上所述,NL63冠状病毒充分利用木瓜样蛋白酶PLP2-TM的多样化功能,负调控天然免疫反应。值得注意的是,NL63编码多功能木瓜样蛋白酶的每种活性之间既相互独立又相互联系,共同维持病毒的复制和释放。本研究对阐明人类新发冠状病毒致病机理和研发抗病毒药物具有重要参考价值。
The immune system includes the innate immune system and adaptive immune system. The innate immune system which is responsible for the defense against microbial pathogens, is a nonspecific way of immunity response. Immunity response is usually started in the early period when the pathogens infected. Viral infections are sensed by pattern-recognition receptors (PRRs) of the innate immune system that recognize pathogen-associated molecular patterns (PAMPs) (RNA, bacterial) and then trigger antiviral response. The activated PRRs recruit different adaptor proteins starting signaling transduction. Most signal proteins are phosphorylated and ubiquitinated to activate the kinase complex (IKK?-IKK?-IKK? and TBK1/IKK? complexes),which can phosphorylate the transcription factors (NF-?B, IRF7, IRF3), which coordinately regulate the production of inflammation cytokines and IFN-?.
Coronaviruses are enveloped viruses with large RNA genomes. To date, five human coronaviruses (HCoV) have been identified as human pathogens. They are HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63 and HCoV-HKU1. After SARS era, researchers have identified two additional HCoV, HCoV-NL63 and CoV-HKU1. HCoV-NL63 was first reported by Van der Hoek L, an Netherlands researcher, which was later found to be associated with croup in young children. Epidemiological studies from several countries revealed that HCoV-NL63 is worldwide spread.
The interaction between virus and host determin the athogenicity and immunocharacterization. The innate immune response is activated once a foreign pathogen is detected in the host. Many viruses interfer or modulate the innate immune response to escape from the defense system. It is reported that more and more viruses encode proteins that block innate immune response. For example, Picornavirus 3C protease is essential for processing the replicase polyprotein and inactivating NF-?B and RIG-I. Coronavirus NL63 (HCoV-NL63), has two papain-like protease(PLP) core domains, PLP1 and PLP2, in nonstructural protein nsp3. We have previously demonstrated that only the core domain of PLP2, but not PLP1, has in vivo DUB activity. As an IFN antagonist, PLP2 interactes with RIG-I and STING (also called MITA / ERIS) of IFN induction pathway, and induces the deubiquitination of RIG-I and ERIS. We found that DUB activity of PLP2-TM is not dependent on its catalytic activity. Nowadays the mechanism that PLP of Human Coronavirus NL63 regulate host antiviral innate immune responses, and the difference between soluble PLP and TM-binding PLP is still unclear. So we researched on the function of TM-binding PLP.
The progress as follows:
1. Cellular location of PLP of Human Coronavirus NL63
(1) Constructs of PLP-TM and their catalytic activity mutants. We used of Molecular colone tecnology and site-directed mutagenesis protocol to colone PLP-TM and its catalytical mutants ( PLP1-TM C1062A, PLP1-TM H1212A, PLP1-TM D1225A, PLP2-TM C1678A, PLP2-TM H1836A, PLP2-TM D1489A ).
(2) We identified the location of PLP-TM and PLP encoded by nsp3 through immunofluorescence assay. Meawhile, we detected the expression of the PLP and the mutants through western blot assay.
2. Biological Characteristics of PLP of Human Coronavirus NL63
(1) First of all, we identified the DUB activity of PLP by DUB detecting assay.We proved that PLP has an TM-dependent DUB activity. However, the DUB activity of PLP2-TM is slightly reduced when the catalytic sites of C1678 and H1836 were mutated. It is suggested that the DUB activity of PLP is not depend on its catalytic residues.
(2) Using the DUB and deISGylation detecting assay, we researched on the substrate specificity of PLP-TM. It shows that PLP2-TM exhibits DUB activity toward ubiquitinated branched peptides without any specificity for either Lys48 linkages or Lys63 linkages. Only PLP2 and PLP2-TM has deISGylation activity, but PLP has an TM–dependent deSUMOylation activity. The results indicated the importance of TM dormain for DUB activity of PLP.
3. IFN antagonist activity of PLP-TM
(1) PLP1-TM and PLP2-TM are responsible for the inhibition of both RIG-I or Sendai virus induction of IFN?/? expression by dual luciferase activity assay. It was proved that PLP1-TM and PLP2-TM are IFN antagonists, and PLP2-TM has a stronger IFN antagonist activity than PLP1-TM.
(2) The IFN antagonist activity of PLP2-TM was checked. We proved that PLP2-TM negatively regulated the induction of IFN in an dose dependent manner, and the protease mutants can also inhibit the expression of IFN with an does dependent activity. It was concluded that the IFN antagonist activity of PLP2-TM is not dependent on its catalytic activity.
4. Mechanism of PLP2-TM negative regulation of host antiviral innate immune response.
(1) Firstly, we detected the IFN? reporter luciferase activity activated by several important signal proteins, co-transfected with PLP2-TM. The data indicated that PLP2-TM can inhibit induction of IFN activated by RIG-I/IPS-1/TRAF3/TBK1/IRF3. Secondly, PLP2-TM deubiquitinates the ubiquitination or ISGylation of RIG-I/ TRAF3/ TBK1/IRF3 detected by coimmunoprecipitation.
(2) Firstly, we researched on upstream and downstream proteins of STING, a new interferon stimulator. We found that PLP2-TM destructs the interaction between STING and IPS-1 not TRAF3 or TBK1. Secondly, interaction between STING and the kinase IKK?/TBK1, it was suggested that PLP2-TM interrupts the interaction between STING and IKK? not TBK1. The results proved that PLP2-TM inhibits the signal transimmited from STING to kinase to suppress the activation of IRF3.
(3) Firstly, we detected the Ubiquitination and ISGylation of STING, and found that PLP2 can deubiquitinate the ubiquitination of RIG-I/STING. Secondly, dimerization is important for STING to activate IFN signaling. We observed the dimerization of STING, and the data shows that PLP2-TM inhibits the dimerization of STING, suppressing the antiviral innate immune responses.
The research provided of the broad evidence showing the mechanism how PLP2-TM negatively regulate the host antiviral innate immune response. Beside the proteolytic processing activity toward pp1a(1ab), we have demonstrated that PLP2-TM of HCoV-NL63 has an in vivo TM-dependent deubiquitinase (DUB) activity, as well as an IFN antagonist. On one hand, PLP2-TM disrupts the ubiquitination of important signal proteins. On the other hand, PLP2-TM inhibits the interacton between STING and IPS-1 or IKK? to supress the induction of IFN. The most important thing is that we first found that PLP2-TM disrupts the dimerization of STING to suppress the translation of IFN. This sdudy is valuable for the research of host antiviral innate immune response. Overall, these results definitely demonstrated that PLP2-TM actes as an multiple function protease to inhibit host antiviral innate immune response. All the functions of PLP2-TM actions separately but congenerously to maintain the replication and generation of virus. The studies will provide the biological significance of DUB of coronavirus PLP2-TM in virus replication and pathogenesis.
冠状病毒是一类拥有庞大RNA基因组的包膜病毒。至今发现的人类冠状病毒包括HCoV-229E、HCoV-OC43、SARS-CoV、HCoV-NL63和HcoV-HKU1五种,其中NL63和HKU1是继SARS冠状病毒后出现的两种人类新发冠状病毒。NL63冠状病毒是2004年由荷兰学者Van der Hoek L首先报道的一种单股正链RNA病毒,呈世界性分布,流行病学检测发现,NL63冠状病毒在儿童和成人(特别是儿童)呼吸道感染中的阳性检测率高,已成为人类呼吸道感染的主要新发病原之一。
冠状病毒与宿主间的相互作用决定了其致病性和免疫特性。冠状病毒感染宿主后会立即启动抗病毒天然免疫反应,病毒为了躲避宿主机体复杂的防御体系,必然对宿主细胞内的信号转导进行干预和调节。近年来,越来越多的研究表明,病毒通过编码多种功能蛋白调控宿主的抗病毒天然免疫反应。例如,小核糖核酸病毒编码的3C蛋白酶一方面剪切复制聚合酶前体,另一方面抑制RIG-I和NF-?B的活化。我们前期研究表明,NL63冠状病毒nsp3编码的木瓜样蛋白酶(PLP)包括两个核心结构域PLP1和PLP2。通过体内DUB活性分析发现,只有PLP2核心结构域具有DUB活性,而PLP1则没有。PLP2作为一种干扰素拮抗剂,通过去除RIG-I和STING的泛素化修饰,负调节干扰素表达,而且PLP2的DUB活性与其干扰素拮抗作用是相互独立的。由于编码PLP2的nsp3位于细胞内质网(ER)膜上,因此NL63冠状病毒nsp3编码的木瓜样蛋白酶应该是膜定位的。目前对于NL63冠状病毒编码的木瓜样蛋白酶(PLP)调控宿主的抗病毒天然免疫反应机制,以及可溶性木瓜样蛋白酶(PLP2)与膜定位的木瓜样蛋白酶(PLP2-TM)之间的差异性尚不清楚。围绕这一问题我们针对NL63冠状病毒编码的膜定位木瓜样蛋白酶(PLP-TM)进行更加深入的研究,主要结果如下:
一. NL63冠状病毒编码的木瓜样蛋白酶(PLP)在细胞内的定位研究
(一)利用分子克隆及基因定点突变技术,分别构建PLP1-TM, PLP2-TM的重组体及蛋白酶活性催化位点突变体(PLP1-TM C1062A, PLP1-TM H1212A, PLP1-TM D1225A, PLP2-TM C1678A, PLP2-TM H1836A, PLP2-TM D1489A)。
(二)对NL63 PLP蛋白酶进行进行免疫荧光染色,确定了nsp3编码的可溶性PLP和膜定位的PLP-TM在细胞的定位情况。结果表明膜定位前后蛋白表达位置发生改变,PLP均匀的分布于细胞质中,而PLP-TM则集中分布于核周内质网上。同时,我们通过western blot检测到PLP-TM及酶活性突变体的表达条带。
二. PLP-TM的生物学特性研究
(一)利用DUB检测技术对NL63 PLP的去泛素化活性进行分析,发现PLP1-TM和PLP2-TM具有明显的DUB活性,证实PLP具有TM依赖的DUB活性;同时,PLP2-TM的蛋白酶催化活性位点(C, H, D)突变后,其DUB活性降低,其中C1678A和H1836的对野生型PLP2-TM的DUB活性影响较大,而D1849A没有影响。说明PLP2-TM的DUB活性不完全依赖其蛋白酶的催化活性。
(二)利用DUB和去ISG检测技术对NL63 PLP的底物特异性进行研究,发现PLP2-TM对Ub-K48和Ub-K63连接的多聚泛素化没有表现出特异性;其次,以类泛素分子(SUMO和ISG15)为作用底物,发现PLP2和PLP2-TM均具有去ISG活性,然而只有膜定位的PLP(PLP1-TM和PLP2-TM)具有去SUMO活性,说明膜定位对于NL63冠状病毒编码木瓜样蛋白酶的功能发挥具有重要作用。
三. PLP-TM负调控宿主干扰素表达活性研究
(一)利用双荧光素酶报告基因检测技术对PLP-TM干扰素拮抗活性进行分析,发现PLP1-TM和PLP2-TM可以下调外界条件(仙台病毒,RIG-IN等)刺激下I型干扰素的表达。说明膜定位木瓜样蛋白酶(PLP-TM)是一种干扰素拮抗剂而且PLP2-TM的拮抗活性强于PLP1-TM。
(二)对PLP2-TM干扰拮抗活性的特异性进行研究,证实PLP2-TM负调节干扰素表达的作用具有剂量依赖性,而且PLP2-TM的三种蛋白酶活性突变体(PLP2-TM C1678A, PLP2-TM H1836A, PLP2-TM D1489A)均能抑制干扰素产生,且存在剂量效应。说明PLP2-TM的干扰素拮抗活性是独立于蛋白酶活性之外的一种特异性功能。
四. PLP2-TM负调控宿主抗病毒天然免疫反应分子机制研究
(一)首先,利用干扰素表达通路中重要调节蛋白激活干扰素表达,同时瞬转PLP2-TM后检测PLP2-TM对信号蛋白介导的干扰素表达通路的调节作用,结果显示PLP2-TM及其突变体明显抑制由RIG-I/TRAF3/TBK1/IRF3激活的干扰素的产生;说明PLP2-TM干扰素拮抗活性不依赖其蛋白酶催化活性;其次,通过免疫共沉淀技术分析,发现PLP2-TM通过去除RIG-I/TRAF3/TBK1/IRF3的泛素化或ISG化修饰负调控干扰素表达通路。
(二)首先,利用免疫共沉淀技术,对最新发现的干扰素刺激因子STING上下游信号蛋白之间的相互作用进行检测,发现PLP2-TM能够破坏STING与IPS-1间的相互作用,但是不破坏STING与TRAF3, STING与TBK1, IPS-1与TRAF3间的相互作用。说明PLP2-TM阻断了信号由IPS-1向STING的流动,以及STING向下游蛋白分子的传递;其次,我们对STING与蛋白激酶间的相互作用进行检测,发现PLP2-TM能够破坏STING与IKK?间的相互作用,但是PLP2-TM不能破坏STING与TBK1间以及TBK1与IRF3间的相互作用,说明PLP2-TM特异性的阻止了STING募集蛋白激酶IKK?至IPS-1,抑制IRF3的活化。
(三)首先,利用免疫共沉淀技术对STING的泛素化及ISG化进行检测,发现PLP2-TM能够破坏STING的泛素化修饰和ISG化修饰;其次,二聚化是STING激活干扰素表达的一个必要条件,而且STING的泛素化和ISG化是在其二聚化基础上进行的,所用利用不同标签的STING进行IP分析,发现PLP2-TM能够破坏STING的二聚化从而抑制IRF3介导的干扰素表达通路。
本课题研究从多方面多角度阐明了PLP2-TM调控宿主抗病毒天然免疫反应机制。实验阐明了NL63编码的木瓜样蛋白酶(PLP)是一种多功能蛋白酶,除了经典的蛋白酶水解活性,还具有TM依赖的去泛素化酶(DUB)活性,同时又作为一种高效的干扰素拮抗剂负调控宿主的抗病毒天然免疫反应。NL63编码PLP2-TM调控宿主抗病毒天然免疫反应机制研究表明,一方面,PLP2-TM通过DUB活性,去除信号通路中重要调节蛋白的泛素化活性,负调控干扰素表达;另一方面,PLP2-TM能够破坏最新发现的干扰素刺激因子STING与IPS-1间, STING与IKK?间的相互作用,阻止信号由STING流向TRAF3和IKK?,从而阻碍了转录因子IRF3的活化,最终抑制I型干扰素的表达。重要的是,我们首次证实PLP2-TM通过破坏STING的二聚化抑制STING的干扰素激活作用,为病毒调控宿主抗病毒天然免疫反应机制研究提供有力参考资料。综上所述,NL63冠状病毒充分利用木瓜样蛋白酶PLP2-TM的多样化功能,负调控天然免疫反应。值得注意的是,NL63编码多功能木瓜样蛋白酶的每种活性之间既相互独立又相互联系,共同维持病毒的复制和释放。本研究对阐明人类新发冠状病毒致病机理和研发抗病毒药物具有重要参考价值。
The immune system includes the innate immune system and adaptive immune system. The innate immune system which is responsible for the defense against microbial pathogens, is a nonspecific way of immunity response. Immunity response is usually started in the early period when the pathogens infected. Viral infections are sensed by pattern-recognition receptors (PRRs) of the innate immune system that recognize pathogen-associated molecular patterns (PAMPs) (RNA, bacterial) and then trigger antiviral response. The activated PRRs recruit different adaptor proteins starting signaling transduction. Most signal proteins are phosphorylated and ubiquitinated to activate the kinase complex (IKK?-IKK?-IKK? and TBK1/IKK? complexes),which can phosphorylate the transcription factors (NF-?B, IRF7, IRF3), which coordinately regulate the production of inflammation cytokines and IFN-?.
Coronaviruses are enveloped viruses with large RNA genomes. To date, five human coronaviruses (HCoV) have been identified as human pathogens. They are HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63 and HCoV-HKU1. After SARS era, researchers have identified two additional HCoV, HCoV-NL63 and CoV-HKU1. HCoV-NL63 was first reported by Van der Hoek L, an Netherlands researcher, which was later found to be associated with croup in young children. Epidemiological studies from several countries revealed that HCoV-NL63 is worldwide spread.
The interaction between virus and host determin the athogenicity and immunocharacterization. The innate immune response is activated once a foreign pathogen is detected in the host. Many viruses interfer or modulate the innate immune response to escape from the defense system. It is reported that more and more viruses encode proteins that block innate immune response. For example, Picornavirus 3C protease is essential for processing the replicase polyprotein and inactivating NF-?B and RIG-I. Coronavirus NL63 (HCoV-NL63), has two papain-like protease(PLP) core domains, PLP1 and PLP2, in nonstructural protein nsp3. We have previously demonstrated that only the core domain of PLP2, but not PLP1, has in vivo DUB activity. As an IFN antagonist, PLP2 interactes with RIG-I and STING (also called MITA / ERIS) of IFN induction pathway, and induces the deubiquitination of RIG-I and ERIS. We found that DUB activity of PLP2-TM is not dependent on its catalytic activity. Nowadays the mechanism that PLP of Human Coronavirus NL63 regulate host antiviral innate immune responses, and the difference between soluble PLP and TM-binding PLP is still unclear. So we researched on the function of TM-binding PLP.
The progress as follows:
1. Cellular location of PLP of Human Coronavirus NL63
(1) Constructs of PLP-TM and their catalytic activity mutants. We used of Molecular colone tecnology and site-directed mutagenesis protocol to colone PLP-TM and its catalytical mutants ( PLP1-TM C1062A, PLP1-TM H1212A, PLP1-TM D1225A, PLP2-TM C1678A, PLP2-TM H1836A, PLP2-TM D1489A ).
(2) We identified the location of PLP-TM and PLP encoded by nsp3 through immunofluorescence assay. Meawhile, we detected the expression of the PLP and the mutants through western blot assay.
2. Biological Characteristics of PLP of Human Coronavirus NL63
(1) First of all, we identified the DUB activity of PLP by DUB detecting assay.We proved that PLP has an TM-dependent DUB activity. However, the DUB activity of PLP2-TM is slightly reduced when the catalytic sites of C1678 and H1836 were mutated. It is suggested that the DUB activity of PLP is not depend on its catalytic residues.
(2) Using the DUB and deISGylation detecting assay, we researched on the substrate specificity of PLP-TM. It shows that PLP2-TM exhibits DUB activity toward ubiquitinated branched peptides without any specificity for either Lys48 linkages or Lys63 linkages. Only PLP2 and PLP2-TM has deISGylation activity, but PLP has an TM–dependent deSUMOylation activity. The results indicated the importance of TM dormain for DUB activity of PLP.
3. IFN antagonist activity of PLP-TM
(1) PLP1-TM and PLP2-TM are responsible for the inhibition of both RIG-I or Sendai virus induction of IFN?/? expression by dual luciferase activity assay. It was proved that PLP1-TM and PLP2-TM are IFN antagonists, and PLP2-TM has a stronger IFN antagonist activity than PLP1-TM.
(2) The IFN antagonist activity of PLP2-TM was checked. We proved that PLP2-TM negatively regulated the induction of IFN in an dose dependent manner, and the protease mutants can also inhibit the expression of IFN with an does dependent activity. It was concluded that the IFN antagonist activity of PLP2-TM is not dependent on its catalytic activity.
4. Mechanism of PLP2-TM negative regulation of host antiviral innate immune response.
(1) Firstly, we detected the IFN? reporter luciferase activity activated by several important signal proteins, co-transfected with PLP2-TM. The data indicated that PLP2-TM can inhibit induction of IFN activated by RIG-I/IPS-1/TRAF3/TBK1/IRF3. Secondly, PLP2-TM deubiquitinates the ubiquitination or ISGylation of RIG-I/ TRAF3/ TBK1/IRF3 detected by coimmunoprecipitation.
(2) Firstly, we researched on upstream and downstream proteins of STING, a new interferon stimulator. We found that PLP2-TM destructs the interaction between STING and IPS-1 not TRAF3 or TBK1. Secondly, interaction between STING and the kinase IKK?/TBK1, it was suggested that PLP2-TM interrupts the interaction between STING and IKK? not TBK1. The results proved that PLP2-TM inhibits the signal transimmited from STING to kinase to suppress the activation of IRF3.
(3) Firstly, we detected the Ubiquitination and ISGylation of STING, and found that PLP2 can deubiquitinate the ubiquitination of RIG-I/STING. Secondly, dimerization is important for STING to activate IFN signaling. We observed the dimerization of STING, and the data shows that PLP2-TM inhibits the dimerization of STING, suppressing the antiviral innate immune responses.
The research provided of the broad evidence showing the mechanism how PLP2-TM negatively regulate the host antiviral innate immune response. Beside the proteolytic processing activity toward pp1a(1ab), we have demonstrated that PLP2-TM of HCoV-NL63 has an in vivo TM-dependent deubiquitinase (DUB) activity, as well as an IFN antagonist. On one hand, PLP2-TM disrupts the ubiquitination of important signal proteins. On the other hand, PLP2-TM inhibits the interacton between STING and IPS-1 or IKK? to supress the induction of IFN. The most important thing is that we first found that PLP2-TM disrupts the dimerization of STING to suppress the translation of IFN. This sdudy is valuable for the research of host antiviral innate immune response. Overall, these results definitely demonstrated that PLP2-TM actes as an multiple function protease to inhibit host antiviral innate immune response. All the functions of PLP2-TM actions separately but congenerously to maintain the replication and generation of virus. The studies will provide the biological significance of DUB of coronavirus PLP2-TM in virus replication and pathogenesis.
引文
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