Functional Analysis Of Papain-like Protease Of Sars-cov

The genome of severe acute respiratory syndrome coronavirus (SARS-CoV) is a positive-sense, single-stranded,29.7kb RNA. The first 2/3 of the genome of SARS-CoV is translated into two large replicase polyproteins called as ppla and pplab. Papain-like protease (PLpro) and 3C-like protease domains present within these polyproteins direct their processing into 16 nonstructural proteins (nsp1-16) that assemble to generate a multifunctional, membrane-associated replicase complex. This complex is responsible for the copy of virus genome and transcription and translation of structural genes. Unlike other coronaviruses that encode two different PLPs(PLP1 and PLP2), SARS-CoV encodes only one PLP domain called PLpro. SARS PLpro recognizes the consensus cleavage sequence LXGG between nsp1-2, nsp2-3, nsp3-4 of the amino terminus of pp1a(1ab) to release nspl, nsp2 and nsp3. Recently, the functions of PLpro of SARS coronavirus is becoming a research focus in coronavirus molecular Biology.SARS PLpro is encoded by nsp3. nsp3 has been annotated as a multidomain protein consisting of a minimum of seven domains. It encodes a transmembrane domain (TM) after PLpro, which locate PLpro on the ER membrane. And there are two small domains of NAB-G2M between PLpro and TM. The PLpro monomer consists of four distinct domains, three of which form an extended right-hand architecture with distinct palm, thumb, and finger domains; the first 62 aminos form an independent N-terminal domain, termed the Ubl domain. In this study, we first constructed the recombination plasmids of PLpro with tansmembrane and PLpro delete N-terminal Ubl domain or NAB-G2M domain. Secondly, the Site-directed mutagenesis was performed to change the catalytic sites (Cys-His-Asp) of PLpro to Ala.We found that the crystal structure of PLpro is significant matched with a cellular deubiquitinase (DUB) HAUSP with the analyzed by Bioinformation method. So, we predict that the PLpro has a potential DUB activity. In order to experimentally test our previous prediction, HEK293T cells were co-transfected with HA-tagged ubiquitin (or K48-linked, K63-linked ubiquitin) and PLpro, constructs or mutants. We found that PLpro and constructs both possesses Deubiquiting activity in vivo, suggesting that the Ubl and NAB-G2M domains are dispensable for the Deubiquiting activity. But the Deubiquitinaion of SARS-CoV PLpro was dependent on Cys-His-Asp catalytic residues.Innate antiviral interferon response can be triggered when virus infection. Viral nucleic acids comprising viral genomes or generated during viral replication present major PAMPs that can be recognized by two different classes of PRRs, the membranebound Toll-like receptors (TLRs) or intracellular receptor retinoic acidinducible gene I (RIG-Ⅰ). Upon engagement of their respective ligands, these PRRs recruit different adaptor molecules, relaying signals to downstream kinases that activate IFN regulatory factor3 (IRF3), nuclear factorκB (NF-κB), transcription factors that coordinately up regulate IFNP transcription and expression. And then, IFN-βbinds to the IFNα/βreceptor (IFNAR), IFNARs trigger the activation of downstream signaling and initiates the transcription of several interferon stimulated genes (ISGs). The ISGs inhibit different stages of virus replication and elicit an anti-viral state in the host. In this study, PLpro, constructs or mutants were co-transfected with IFNβ-Luc and pRL-TK reporters into HEK293T cells. At 24 hours post transfection, the cells were either mock treated or treated with 100HAU/ml Sendai virus for 16 h to stimulate IFNβpathway. Or the RIG-IN, IPS-1, IKKεor IRF3 was used to stimulate IFNβinduction. Cell lysates were harvested and assayed for luciferase activity via the Dual Luciferase Reporter Assay. We demonstrated that the PLpro, contructs or mutants were all interferon antagonist. PLpro and the catalytic mutants inhibit IFNβinduction in a dose-dependent manner.This paper further explores the molecular mechanisms that PLpro inhibits the IFNβpathway. First, to determine if PLpro associate with TRAF3/TBK1/IKKs/ERIS, co-immunoprecipitation experiments were performed. HEK 293T cells were co-transfected with plasmid DNA expressing a Flag-tagged version of TRAF3/TBK1/IKKε/ERIS in the presence of PLpro-TM and cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The results show that PLpro was detected in association with TRAF3/TBK1/IKKε/ERIS. Then, we demonstrated that PLpro-TM disrupts TRAF3 interaction with IKKs, TRAF3 with TBK1 and ERIS with IRF3. It suggested that PLpro inhibit the IFNβexpression by blocking the TRAF3 complex. In addition, Protein ubiquitylation has emerged as a key mechanism that regulates immune responses. Much like phosphorylation, ubiquitylation is a reversible covalent modification that regulates the stability, activity and localization of target proteins. Many pathway molecules (RIG-Ⅰ, ERIS, TRAF3 and IRF3) can be modified by ubiquitin. RIG-Ⅰand TRAF3 are both modified by K63 poly-ubiquitin. HEK 293T cells were transfected with HA-Ub and epitope-tagged versions of either RIG-Ⅰ, ERIS, TRAF3 or IRF3 and cell lysates were subjected to immunoblotting to determine the status of ubiquitination of each target. The results indicate that the PLpro-TM is capable of removing ubiquitin from molecules that play key signaling roles in the induction of interferon.Summarize all the experiments. We get the following conclusions:First, SARS PLpro is a novel DUB encoded by virus. Second, SARS PLpro inhibits IFN response of host antiviral innate immunity. Thirdly, SARS PLpro suppressing of IFNβpathway via blocking the form of TRAF3 complex and removing ubiquitin from molecules that play key signaling roles in the interferon pathway. Our study expounds the new mechanisms of inhibiting innate immunity pathway by SARS PLpro, which provide the theoretical base for the development of new antiviral drugs.