Regulation Of Antiviral Innate Immune Responses By Papain-like Proteases Of Human Coronavirus NL63

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.