Investigation Of The Mechanism Of Du-cGAS-mediated Antiviral Innate Immune Response

Cyclic GMP-AMP synthase(cGAS)is an important protein involved in the signal transduction pathway of pattern recognition receptors,playing a key role in cell apoptosis and immune regulation.Since its discovery in2012,the structure and function of cGAS have become a hot topic in immunology research.The function of cGAS is to recognize foreign DNA and initiate an antiviral immune response.cGAS can bind to a variety of viral DNAs,including cytomegalovirus(CMV),Kaposi’s sarcomaassociated herpesvirus(KSHV),adenovirus,HSV,and cowpox virus(CPV),etc.The cGAS-STING pathway is typically activated by DNA viruses,but it can also initiate an immune response to RNA virus infection.However,the functional response of duck cGAS(du-cGAS)to viral infection is still unclear.This study aims to explore the natural immune response mechanism of cGAS in DEF cells,laying a foundation for further research on DEF’s antiviral innate immunity.The du-cGAS gene was cloned using DEF(SPDC-CCL141)cells’ m RNA as a template and prepared using prokaryotic expression technology and protein affinity purification technology to generate du-cGAS polyclonal antibodies.Subsequently,SBDSV-M15,NDRV-NP03,and DAd V-B2-BG61 were used to infect cGAS-deficient cells and cGAS-overexpressing cells.The differences in virus proliferation and changes in the transcription of host cell interferon signaling pathway-related genes were detected through q RT-PCR,Western blot,and laser confocal microscopy.The du-cGAS gene was analyzed using relevant bioinformatics software,and the amino acid sequences of the NTase,Mab21,and Zinc-Ribbons domains were expressed in segments through segmentation analysis.The antiviral activity of du-cGAS was explored by comparing the differences in virus proliferation and the expression levels of interferon signaling pathway-related genes.The following conclusions were drawn:(1)The open reading frame(ORF)region of the du-cGAS gene is 1296 bp,encoding 432 amino acids(aa),and is located on the same evolutionary branch as its chicken source cGAS.(2)Specific sg RNA designed to knock out endogenous du-cGAS significantly promoted the replication of DNA viruses(Duck adenovirus B2,DAd V-B2,and Duck short beak and dwarfism virus,SBDSV).However,the replication of double-stranded RNA virus NDRV was not significantly affected.(3)In the du-cGAS-deficient DEF cell line,DNA virus infection could not stimulate the upregulation of type I interferon(IFNs)and important interferon-stimulated genes(ISGs)m RNA expression,but RNA virus infection could still upregulate the cell IFNs and ISGs gene expression normally.(4)Overexpression of du-cGAS significantly upregulated the transcription of IFNs and important ISGs and inhibited the replication of DAd V-B2,SBDSV,and NDRV in DEF cell lines,showing a broad-spectrum antiviral effect.(5)The study found that truncation of the cGAS protein’s N-terminus by 68 amino acids did not impair du-cGAS’s antiviral function,and overexpression of NTase Core,C-Domain(Mab21),or Zinc-Ribbons domain independently did not have an antiviral effect.The above results indicate that du-cGAS is an important component of the innate immune system in ducks,with broad antiviral activity,and provides a beneficial strategy for controlling viral diseases in waterfowl.