| As a host restriction factor,SAMHD1(SAM domain and HD domain containing protein 1)has become a research hotspot in the field of molecular biology due to its unique antiviral function.In mammals,SAMHD1 achieves antiviral function by hydrolyzing dNTPs in cells,depleting the raw materials required for retroviral replication.In addition to inhibiting retroviral replication,SAMHD1 is also participated in the STING-mediated apoptotic pathway,and as a host restriction factor,the knockout of SAMHD1 will result in a serious autoimmune disease.Although SAMHD1 has been extensively studied in mammals,it has been reported very rarely in fish.This study explored the transcriptional regulation mechanism of fish SAMHD1 and its function in the innate immune signaling pathway,and also explored the role of fish SAMHD1 in inhibiting GCRV virus.In this study,the full-length cDNA(2792 bp)of CiSAMHD1 was firstly cloned by homologous cloning and RACE technology,including 5 'UTR of 75 bp,3' UTR of 833 bp and 1884 bp open reading frame(ORF),which encoded a protein containing 628 amino acids.Protein structure analysis indicated that CiSAMHD1 has the same SAM domain(nucleic acid binding site)and HD domain as mammalian SAMHD1,and has the highest homology with fish SAMHD1.In order to study the expression characteristics of grass carp SAMHD1 in CIK cells,exogenous or endogenous polyI:C,B-DNA and Z-DNA stimulation were performed on CIK cells respectively,and the mRNA and protein levels of grass carp SAMHD1 were detected at different time points : Grass carp SAMHD1 is only up-regulated in response to stimulation by endogenous nucleic acids(poly I:C,B-DNA and Z-DNA).Furthermore,recombinant grass carp IFN can also induce the expression of SAMHD1 in CIK cells.This suggests that the expression of CiSAMHD1 may be regulated by multiple signaling pathways.To further study the transcriptional regulation mechanism of CiSAMHD1,the promoter sequence of CiSAMHD1 was found in the grass carp genome database,and the promoter elements were predicted online.It was found that the CiSAMHD1 promoter contains IRF1,IRF3,ISGF3,NF-kappaB,which are potential binding elements.Gel mobility shift assays showed that IRF1,IRF3,IRF9 and p65 were able to bind to the promoter of grass carp SAMHD1.qRT-PCR and dual luciferase reporter system experiments demonstrated that overexpression of IRF3 and IRF9 in CIK cells up-regulated the transcription level of SAMHD1,whereas overexpression of IRF1 and p65 did not.The same conclusion was confirmed in the experiments of knocking down IRF1,IRF3,IRF9,and p65.The first part of the study indicated that the transcription of CiSAMHD1 was regulated by IRF3 and IRF9,while the second part continued to explore the function of CiSAMHD1 in the innate immune pathway.confocal microscopic examination and nuclear cytoplasmic extraction suggested that CiSAMHD1 were translocated from the nucleus to the cytoplasm under the stimulation of endogenous polyI:C,B-DNA and Z-DNA.In order to further explore the biological significance of nucleation,nucleic acid pull-down was found that SAMHD1 can bind to ISD-PS(RNA analog),B-DNA,and Z-DNA.Western blot,qRT-PCR and dual luciferase reporter system results showed that overexpression of SAMHD1 in CIK cells up-regulated IFN expression,while knockdown of SAMHD1 inhibited IFN expression.To further explore the mechanism by which SAMHD1 up-regulates IFN,co-IP experiments revealed that SAMHD1 interacts with STING and simultaneously overexpresses SAMHD1 and STING in grass carp CIK cells,and mRNA levels of IFN increase significantly.To sum up,grass carp SAMHD1 is up-regulated by IRF3 and IRF9 in response to endogenous polyI:C,B-DNA,Z-DNA and recombinant IFN,respectively,Subsequently,SAMHD1 is transferred from the nuclear part of the cell to the cytoplasm and bound to the endogenous nucleic acid,recruiting STING and interacting with STING.The STING-mediated signaling pathway ultimately up-regulates IFN expression.Moreover,grass carp SAMHD1 can inhibit the amplification of GCRV virus in CIK cells. |
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