Mechanisms And Regulations Of Protein Kinase NEK6-Mediated Cellular Antiviral Signaling And Antiviral Activity Research Of DNAzyme

Antivral innate immunity system establishs the first defensive line against viruses infection. Detection of viral components by immune cells activates intracellular antiviral signaling cascades, leading to the secretion of type I IFNs, proinflammatory cytokines and chemokines. Type I IFNs activate intracellular signaling pathways via type ⅠIFN receptors and regulate the expression of a set of genes.The IFN-inducible genes, such as protein kinase R and2’5’-oligoadenylate synthase,are involved in eliminating viral components from infected cells,inducing apoptosis of infected cells and conferring resistance to viral infection on uninfected cells. Proinflammatory cytokines and chemokines are also critical for eliminating virus infection by provoking inflammation and recruiting innate and acquired immune cells. Costimulatory molecules are essential for the activation of T cells,leading to maturation of acquired immune system.The innate immune system recognizes pathogen-associated molecular patterns(PAMPs) from viruses via a limited number of germline encoded pattern-recgnition receptors(PRRs).The PRRs involving virus recogiton include Toll-like receptors(TLRs), RIG-I-like receptors(RLRs) and cytosolic DNA sensors. Membrance-lacated TLRs activate downstream cellular signal pathway via adpator proteins MyD88or TRIF,leading to the production IFNs and cytokines. Upon viral infection the cytosolic RIG-I-like receptors(RLR) recuit another adpator VISA and form a VISA/TRAFs/TANK/NAP1/SINTBAD complex to activate kinase TBK1and IKKs, which in turn promotor IRF3and NF-κB to transcipt IFN gene. The post translation modifications,such as phosphorylations and ubiquitinations, are very important in cellular life cycle. We speculate there are still some kinaes to be dig out from the genome.We have gotten a new protein kinase-Nek6affecting the production of Type I IFNs from a protein kinases expression library. Overexpressin of Nek6inhbits ISRE,IFN promoter and IFNB1and RNATES expression acivated by SeV, whereas Knowdown of Nek6by siRNA has oppsite effects and suppresses VSV replication. Our biochemical experiments show Nek6promotes activated IRF3degradation via ubiquitnation-dependent manner. We also find Nek6can phosphorylate IRF3at173serine under overexpression condition.Our findings imply Nek6negatively regulates cellular antiviral signaling transduction. Nek6 belongs to NIMA-related kinases family,which are mainly implicated in cell cycle control. We will use antibody sepecificitly recoginzing IRF3173phosphorylated serine to furthe check the phosphorylation status of endogenous IRF3during virus infection and cell cycles,which will fullfill our understanding about the molecular mechnism of Nek6in antiviral innate immune system.Severe acute respiratory syndrome (SARS) outbreaked in2003has been entologically linked to a novel coronavirus named the SARS-CoV.The viral genome is a linear and positive single stranded RNA about30kb in length,containing14open reading frames(ORFs) and highly coservertive untranslated regions at both termini. Currently,there are no approved antiviral drugs that are highly effective against SARS-CoV.Several RNA-cleaving DNAzyems have been reported to cleave their substrate RNA under simulated physiological condition efficiently.Two catalytic motifs,10-23and8-17have been selected from a combinatorial library of DNA sequences. To date,a number of DNAzymes have been designed to inhibit HIV,HPV and influenza virus. In this study we explore the potential use of DNAzymes targeting the SARS5’UTR. In order to examine the efficacy of the desinged DNAzymes,we use a fusion transcript of the5’UTR and GFP gene as a model system.The DNAzyme designed was found to be effective both in vitro and in vivo. We will use SARS subgenome replicon system to check the inhibiting efficacy of DNAzyme on virus replication and screen more DNAzymes targeting at the whole genome of SARS.We will try to improve the activity and stability of DNAzyme by chemical modification,which will pave the way for new class of anti-SARS agent.