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Regulation of Antiviral Innate Immune Responses by Papain-like Proteases of Human Coronavirus NL63

Author: SunLi
Tutor: ChenZhongBin
School: PLA Military Academy of Medical Sciences
Course: Biochemistry and Molecular Biology
Keywords: Coronavirus NL63 Papain -like protease Deubiquitinating enzyme Interferon antagonist STING
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 99
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Abstract

The host's immune system is divided into innate immunity (Innate immunity) and Acquired Immune (Adaptive immunity). Natural immune non-specific form of immune response, based on the recognition and clear pathogens based defensive barrier. The innate immune reaction occurs mainly in the early stage of viral infection and is closely related with the anti-viral infection. After the virus infection, the host will immediately start the natural immune response, detecting various pathogen-associated molecular pattern (PAMP) through the cell surface and cytoplasm of pattern recognition receptors (PRR), such as the viral RNA, such as bacterial cell wall components, starting a series of cell signal transduction cascade. Pattern recognition receptors activated by the same type of interaction with the adapter protein phase, the start signal transduction. Biotinylated modified in the process of signal transduction through phosphorylation and pan-signaling pathway is activated genes induced protein kinase (as of IKK?-Of IKK?-IKK?, And TBK1/IKK? Kinase complex) activation, thereby activating transcription factors (such as NF-? B, IRF7, IRF3,), and ultimately lead to pro-inflammatory cytokines and type I interferon gene transcription, jointly mediated antiviral effect. The coronavirus is a huge RNA genome enveloped viruses. Has found that the human coronavirus HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63 and five HcoV-HKU1, which NL63 and HKU1 Following the SARS coronavirus appearing two human hair coronavirus. NL63 coronavirus is a single-stranded RNA virus was first reported in 2004 by the Dutch scholar Van der Hoek L, was a worldwide distribution, epidemiology detected NL63 coronavirus respiratory tract infections in children and adults (especially children) The positive detection rate, has become a major human respiratory infections the new incidence original one. Coronal interaction between the virus and host its pathogenicity and immune characteristics. Coronaviruses infect host will immediately start antiviral innate immune response the virus to escape the host organism complex defense system will inevitably intervene and adjust the signal transduction within the host cell. In recent years, a growing number of studies show that the virus by encoding a variety of functional protein regulation host antiviral natural immune response. For example, the one hand, the 3C protease encoded picornaviruses Cut Copy polymerase precursor other hand, inhibition of RIG-I and of NF-? B activation. Our previous studies have shown, the of NL63 coronavirus nsp3 coding papaya-like protease (PLP) consists of two core domains PLP1 and PLP2. Vivo by DUB activity analysis showed that only PLP2 core domain with DUB activity, while PLP1 no. PLP2 as an interferon antagonist, by removing the ubiquitination of RIG-I and STING, a negative regulator of interferon expression and PLP2 DUB activity of its interferon antagonism independent. Coded the PLP2 the nsp3 located in the endoplasmic reticulum (ER) membrane NL63 coronavirus nsp3 coding papaya-like protease membrane localization. NL63 coronavirus encoding the papain-like protease (PLP) natural regulation of the anti-viral host immune response mechanism, and a soluble papain-like protease (PLP2) and the membrane localization of the papain-like protease (PLP2-TM) between the differences is not yet clear. Around this problem we NL63 coronavirus encoded membrane localization papaya-like protease (PLP-TM) to conduct a more in-depth study, the main results are as follows:. NL63 coronavirus encoded papaya-like protease (PLP) in the intracellular localization (a) the use of mutation techniques in molecular cloning and gene designated, respectively, to build PLP1-TM PLP2-TM reorganization body and protease activity of the catalytic site mutant body (PLP1-TM C1062A the PLP1-TM H1212A, the PLP1-TM D1225A, PLP2- TM C1678A, PLP2-TM H1836A, PLP2-TM D1489A). (B) NL63 PLP protease immunofluorescence staining to determine the of nsp3 encoding soluble PLP and membrane localization of PLP-TM in the cell's location. The results show that the membrane localization of protein before and after the position change, PLP is uniformly distributed in the cytoplasm, and PLP-TM is concentrated in the Nuclear weeks endoplasmic reticulum. At the same time, we PLP-TM and the expression of the mutant enzyme activity detected by western blot bands. PLP-TM biological characteristics of (a) the use of the DUB detection technology NL63 PLP deubiquitination activity analysis found the the PLP1-TM and PLP2-TM significant DUB activity confirmed PLP TM dependence DUB activity; PLP2-TM the protease of catalytically active sites, (C, H, D) mutant, reduce its DUB activity, wherein greater impact C1678A and H1836 for the DUB activity of the wild-type PLP2-TM, and the D1849A no effect . Description PLP2-TM DUB activity is not completely dependent on the catalytic activity of the protease. (B) the use of the the DUB and go ISG detection technology NL63 PLP substrate specificity studies, found that poly-ubiquitinated PLP2-TM Ub-K48 and Ub-K63 connection did not exhibit specificity; Secondly, the class ubiquitin molecule (SUMO and ISG15) substrates of discovery of PLP2 and a PLP2-TM have to go ISG activity, however, only the membrane localization of PLP (PLP1-TM and PLP2-TM) go to SUMO activity, membrane localization NL63 The coronavirus coding papain-like proteases play an important role. Three. PLP-TM negative regulation of host interferon expression activity of (a) the use of dual luciferase reporter gene assay PLP-TM interferon antagonist activity analysis, discovery the PLP1-TM PLP2-TM can cut external conditions (Sendai virus, RIG-IN) stimulate the expression of type I interferon. Description the membrane localization papaya like protease (PLP-TM) is an interferon antagonist but antagonistic activity of PLP2-TM stronger than PLP1-TM. (B) interference PLP2-TM antagonistic activity of specific research confirmed PLP2-TM negative regulator of interferon expression in a dose-dependent, but the three protease activity of the PLP2-TM mutant body (PLP2-TM C1678A PLP2 -TM H1836A, PLP2-TM D1489A) inhibited the production of interferon, and there is a dose-response. PLP2-TM interferon antagonist activity is independent on the outside of the protease activity, a specific function. The molecular mechanisms of the innate immune response. PLP2-TM a negative regulator of the host antiviral research (a) First of all, the use of interferon the expression pathway important regulatory proteins activate interferon expression, and at the same time instant turn PLP2-TM detected after PLP2-TM protein mediated signal guide interferon expression pathways regulating role results show PLP2-TM and its mutant significantly inhibit the production of interferon by RIG-I/TRAF3/TBK1/IRF3 activated; PLP2-TM interferon antagonist activity does not depend on its protease The catalytic activity; followed by immune coprecipitation technical analysis found PLP2-TM by removing RIG-I/TRAF3/TBK1/IRF3, ubiquitination or ISG modified negative regulation of interferon expression pathway. (B) First, the use of co-immunoprecipitation techniques, the latest discovery of interferon to stimulate interaction between the factor STING on downstream signaling protein detection, PLP2-TM can destroy STING IPS-1 interaction, but not destruction STING TRAF3, STING and TBK1, IPS-1 and the TRAF3 between the interaction. PLP2-TM blocking signal to STING flow from IPS-1, and STING passed downstream protein molecules; Second, we detect interactions between of STING and protein kinase PLP2-TM can destroy STING and IKK ? interaction between PLP2-TM can not destroy the interaction the STING TBK1 between TBK1 and IRF3 between PLP2-TM specificity prevent STING raised protein kinase of IKK? to the IPS-1, inhibit IRF3 activation. (C) First, the use of immune coprecipitation detection technology STING ubiquitination and ISG found PLP2-TM can disrupt the ubiquitination of STING and ISG modification; followed by dimerization STING activate interferon expression a necessary condition, and the STING ubiquitination and ISG dimerize based on IP analysis with the use of different labels STING, STING dimerization of PLP2-TM can destroy thereby inhibiting IRF3 mediated guide interferon expression pathway. This study is clear from various angles PLP2-TM regulation of host antiviral innate immune response mechanism. Experiments to clarify the NL63 encoding papaya-like protease (PLP) is a multifunctional protease deubiquitinating enzyme (DUB), TM-dependent activity of the proteolytic activity in addition to the classic, which also serves as a highly effective interferon antagonists negative natural regulation of host antiviral immune response. The natural immune response mechanisms of regulation of the host antiviral NL63 encoding PLP2-TM show that the one hand, PLP2-TM DUB activity, removal of the signal path, an important regulator of protein ubiquitination activity, expression negative regulation interferon; the other hand, PLP2-TM can destroy the latest discovery of interferon stimulated with IPS-1 Jian factor STING, STING and of IKK? interaction between stop signal by the the STING flow of TRAF3 and IKK?, thus preventing the activation of the transcription factor IRF3 ultimately inhibit I expression of type I interferon. Important, we demonstrated for the first time that PLP2-TM to inhibit STING interferon destruction STING dimerization activation of viral regulation of host antiviral innate immune reaction mechanism studies provide strong references. In summary, and NL63 coronavirus take full advantage of the papaya-like protease PLP2-TM diversification negative regulator of the innate immune response. It is worth noting that, NL63 encoding each of the multi-papain-like protease activity between both mutually independent and mutually contact and common sustain viral replication and release. In this study, to elucidate the pathogenic mechanism of new hair human coronavirus research and development of antiviral drugs has important reference value.

Regulation of Antiviral Innate Immune Responses by Papain-like Proteases of Human Coronavirus NL63

Abstract

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