We performed coimmunoprecipitation experiments to determine which protein is the target of SARS\CoV\2 ORF9b. endosome RNA\sensing pathway of TLR3\TRIF signaling and the adaptor protein of the cytosolic DNA\sensing pathway of O4I2 cGASCSTING signaling, respectively. A mechanistic analysis revealed that this SARS\CoV\2 ORF9b protein interacted with RIG\I, MDA\5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS\CoV\2 ORF9b facilitated the replication of the vesicular stomatitis computer virus. Therefore, the results showed that SARS\CoV\2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS\CoV\2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID\19. gene, can remarkably suppress RIG\I/MDA\5CMAVS, TLR3CTRIF, and cGASCSTING signaling\activated types I and III IFN production by targeting multiple molecules of these innate antiviral pathways. 2.?MATERIALS AND METHODS 2.1. Reagents and antibodies Protein A/G beads were purchased from Santa Cruz Biotechnology, and the anti\Flag magnetic beads were purchased from Bimake. Poly (I:C) and 2?3?\cGAMP were purchased from Invivogen. Rabbit anti\Myc\tag (71D10), rabbit anti\IRF3 (D83B9), rabbit anti\pIRF3 (4D46), rabbit anti\TBK1 (3031S), rabbit anti\pTBK1 (D52C2) were purchased from Cell Signaling Technology. Mouse anti\MAVS was purchased from Santa PRKAR2 Cruz Biotechnology. Mouse anti\actin, mouse anti\V5, and rabbit anti\calnexin antibodies were purchased from proteintech. Mouse anti\Flag M2 antibody was purchased from Sigma\Aldrich. Mouse anti\Myc\tag (9E10) was purchased from Origene. Rabbit anti\GM130 was purchased from Abcam. Rabbit anti\Tom20 antibody was purchased from Abclonal. Mouse anti\HA was purchased from MDL Biotech. 2.2. Constructs and plasmids Plasmids expressing RIG\I, RIG\IN, MDA\5, MAVS, TBK1, IKK, IRF3\5D, TRIF, and STING were cloned into mammalian expression vectors, and the luciferase reporter plasmids including pGL3\IFN\\Luc (IFN\ luciferase reporter) and pGL3\IFN\1\Luc (IFN\1 luciferase reporter) were constructed by inserting the promoter region into pGL3\Basic by standard molecular cloning methods as described in our previous publications. 28 , 29 , 30 pISRE\Luc (the luciferase reporter of ISGs) was purchased from Clontec. The SARS\CoV\2 ORF9b gene was synthesized according to the genome sequence of the SARS\CoV\2 Wuhan\Hu\1 strain (“type”:”entrez-nucleotide”,”attrs”:”text”:”NC_045512.2″,”term_id”:”1798174254″,”term_text”:”NC_045512.2″NC_045512.2) at General Biol. The coding region of the SARS\CoV\2 O4I2 ORF9b gene was amplified using primers list in Table?S1 by polymerase chain reaction (PCR) and cloned into the pCAG mammalian expression vector with a C\terminal Flag\tag. 2.3. Cell culture HEK\293T, HeLa, and Vero E6 cells were obtained from the American Type Culture Collection (ATCC) and managed according to the culture methods provided by the ATCC. All these cells were cultured in Dulbecco’s altered Eagle’s medium (DMEM) with 10% warmth\inactivated fetal bovine serum (FBS) at 37C in a humidified incubator with 5% CO2. 2.4. Transfection The plasmids were transiently transfected into the cells using Lipofectamine 3000 (Invitrogen) or Polyethylenimine Maximum (Polysciences, Inc.) following the manufacturer’s training. Poly (I:C) and 2?3 cGAMP were delivered into cells using Lipofectamine 2000 (Invitrogen) as described previously. 28 2.5. RNA extraction and actual\time quantitative PCR Total RNA O4I2 was extracted with TRIzol reagent (Invitrogen) and then was reverse\transcribed into first\strand cDNA with the HiScript III 1st Strand cDNA Synthesis Kit with gDNA wiper (Vazyme) following the manufacturer’s protocol. The SYBR Green\based RT\qPCR kit UltraSYBR Combination (CWBIO) was used to perform actual\time quantitative PCR (RT\qPCR) assays using primers of each gene (Table?S1) by a Roche LightCycler96 system according to the manufacturer’s instructions. The relative expression of the indicated genes was normalized to the mRNA level of glyceraldehyde 3\phosphate dehydrogenase, one of the internal housekeeping genes in human cells. A comparative and further incubated with the indicated antibodies for 3?h at 4C followed by the addition of protein A/G beads (Santa Cruz), or with anti\Flag magnetic beads (Bimake), anti\Myc magnetic beads.