Studies On The Effect And Mechanism Of DEAD-box RNA Helicase 21 And 10 On PRRSV Replication

Porcine reproductive and respiratory syndrome virus（PRRSV）is an important pathogen that threatens the global swine industry.Due to its highly variable characteristics and complex immune escape mechanisms,existing vaccines cannot provide effective immune protection,leading to difficulty of clinical prevention and control.Therefore,in-depth study of the interaction between PRRSV and the host,and the discovery of the host’s antiviral mechanism and PRRSV’s immune escape strategy are of great significance for the development of new PRRSV vaccines,drug development and prevention and control measures.DEAD-box（DDX）family proteins are a class of adenosine triphosphate（ATP）-dependent RNA helicases,which are widely involved in various life activities such as RNA splicing,transcription,and translation in host cells,and more and more studies have confirmed that DDXs are also involved in innate immune regulation and viral replication,becoming the current research hotspots.At present,there are few studies on DDXs regulating PRRSV replication and its mechanism.In order to systematically study the role of DDXs in regulating PRRSV replication,this study used recombinant PRRSV expressing green fluorescent protein（EGFP）to screen DDXs proteins that could affect PRRSV proliferation,and further studied the DDXs that significantly promoted or inhibited PRRSV proliferation.The main research contents are as follows:1.Screening for potential DDXs family proteins regulating PRRSV proliferationMARC-145 cells were transfected with 40 eukaryotic expression plasmids encoding DDXs proteins,respectively and then infected with recombinant PRRSV with EGFP tags（r HP-PRRSV/SD16/TRS6-EGFP）.The infection of PRRSV was detected by flow cytometry.The results showed that DDX21,DDX4 and DDX27 significantly promoted PRRSV proliferation,while DDX10 and DDX49 significantly inhibited PRRSV proliferation.The DDXs that promoted or inhibited PRRSV proliferation were further confirmed by real-time quantitative RT-PCR and the results were consistent with that of flow cytometry analysis.Taking into account the regulatory effects of the screened DDXs on PRRSV proliferation（promotion or inhibition）and the current international research on these DDXs,we selected DDX21,which significantly promoted PRRSV proliferation,and DDX10,which significantly inhibited PRRSV proliferation,for further study.2.Study on the mechanism of DDX21 promoting PRRSV proliferationTo further determine the effect of DDX21 on promoting PRRSV proliferation,in combination with IFA,Western blot,q RT-PCR and TCID₅₀ analysis,both overexpression and si RNA interference were used to confirm that overexpression of DDX21 significantly promoted PRRSV proliferation,while interference with endogenous DDX21 expression significantly inhibited PRRSV proliferation.Meanwhile,three enzymatic activity-deficient mutants of DDX21 were constructed and evaluated for their effects on PRRSV proliferation.It was found that DDX21 promoted PRRSV proliferation independently of its ATPase activity,RNA unwinding enzyme activity and RNA folding enzyme activity.In parallel to the analysis of DDX21 regulating PRRSV proliferation,the effect of PRRSV infection on DDX21 expression was also analyzed.PRRSV infection was found to significantly upregulate the m RNA and protein expression of DDX21 in a time-and dose-dependent manner.To determine which PRRSV-encoded proteins could interact with DDX21 and affect its expression,a comprehensive analysis of the interaction of PRRSV-encoded proteins with DDX21 was performed by Co-IP assay.The results showed that non-structural proteins nsp1α,nsp1β,nsp4,nsp12 and structural protein N（nucleocapsid protein）encoded by PRRSV interacted with DDX21 and that nsp1βsignificantly up-regulated the m RNA and protein expression of DDX21.By constructing truncated mutants with different structural domains of DDX21,the C-terminus of DDX21 was identified as the key structural domain for its interaction with nsp1β.DDX21 was also found to significantly up-regulate the expression of nsp1α,nsp1β,and N proteins,which are known antagonists of interferon-β（IFN-β）.In addition,we analyzed whether DDX21 affects viral proliferation by regulating the IFN signaling pathway under PRRSV infection conditions and found that knockdown of DDX21 significantly activated the IFN-βsignaling pathway and induced the expression of interferon-stimulated genes（ISGs）.These results suggest that DDX21 can promote PRRSV proliferation through various mechanisms,including down-regulating the expression of IFN-βand ISGs,up-regulating the expression of virus-encoded IFN antagonist proteins,and interacting with viral proteins.3.Study on the mechanism of DDX21 negatively regulating Se V-mediated IFN-βGiven that DDX21 inhibits IFN-βproduction and promotes PRRSV proliferation,the mechanism by which DDX21 regulates IFN production was further investigated.Through overexpression and si RNA interference,it was confirmed that DDX21 negatively regulates Sendai virus（Se V）-induced IFN-βproduction independent of its ATPase activity,RNA unwinding enzyme activity and folding enzyme activity.To exclude the off-target effect of si RNA and other factors,DDX21 knockout HEK-293T cell lines were constructed using CRISPR/Cas9 technology.Comparison Se V-induced IFN-βproduction,ISG expression and the expression of key signaling molecules or transcription factors in the interferon signaling pathway（IKKε,IRF3,p65,STAT1,STAT2）and their phosphorylation levels on DDX21 knockout cells and wild-type cells,we found that knockout of DDX21 significantly upregulated Se V-induced IFN-βproduction,interferon-stimulated genes（ISG15,Mx1,OAS1）m RNA expression and the phosphorylation levels of IKKε,IRF3,p65,STAT1,STAT2.Meanwhile,overexpression of DDX21 on a DDX21 knockout cell line（replenishment experiment）also confirmed that DDX21 negatively regulates Se V-induced IFN-βproduction.To identify the target of DDX21’s negative regulation of IFN-βproduction,the effect of DDX21 on the RLR（RIG-I-like receptor）signaling pathway was analyzed and it was found that overexpression of DDX21 did not affect IFN-βproduction induced by RIG-I,MDA5,IPS-1,TBK-1,IKKεand IRF3,suggesting that the target of DDX21 may be located in RIG-I/MDA5 or their upstream.Knockout,overexpression and Co-IP assays revealed that DDX21 did not affect RIG-I expression nor interacted with RIG-I.RNA pull-down and competitive binding assays revealed that DDX21 competes with RIG-I for binding to double-stranded RNA（ds RNA）and that DDX21 competes with RIG-I for binding to ds RNA mainly through its 217-784 amino acid（aa）region,thereby inhibiting IFN-βproduction.These results suggest that DDX21 inhibits RLR-mediated IFN-βproduction by competing with RIG-I for binding to ds RNA.4.Study on the mechanism of DDX10 inhibiting PRRSV proliferationUnlike DDX21,the flow cytometry screen results showed that DDX10 has the ability to inhibit PRRSV proliferation.To further validate its antiviral effect,overexpression or si RNA interference experiments were performed in MARC-145 cells and i PAM cells（porcine alveolar macrophage cell line）followed by IFA,Western blot and TCID₅₀ assays,The results confirmed that overexpression of DDX10 inhibited PRRSV proliferation,while knockdown DDX10 promoted PRRSV proliferation,indicating that DDX10 inhibited PRRSV proliferation independently of cell type.To investigate whether the inhibition of PRRSV proliferation by DDX10 is related to the regulation of IFN,the effect of DDX10on IFN production was analyzed in HEK-293T and i PAM cells,and it was found that overexpression of DDX10 promoted activation of transcription factors IRF3 and NF-κB and IFN-βproduction,In contrast,knockdown DDX10 inhibited Se V-induced activation of IRF3 and NF-κB,IFN-βproduction and expression of ISGs（ISG15,OAS1,Mx1,IFITM3）,suggesting that DDX10 may inhibit PRRSV proliferation by suppressing induction of IFN production and signaling,and this inhibitory effect was also independent of its ATPase activity and RNA unwinding enzyme activity.Meanwhile,the effect of PRRSV infection on DDX10 expression and subcellular localization was analyzed,and it was found that PRRSV infection caused migration of DDX10 from the nucleus to the cytoplasm and degraded DDX10.To determine the pathway by which PRRSV degrades DDX10,cells were treated with specific drugs that inhibit the apoptotic pathway（Z-VAD-FMK）,the ubiquitin proteasome pathway（MG132）,the autophagic lysosomal pathway（CQ,NH₄Cl）,respectively.The results showed that CQ or NH₄Cl treatment significantly inhibited the degradation of DDX10 by PRRSV,suggesting that PRRSV infection may degrade DDX10through the autophagy-lysosomal pathway.The interaction of PRRSV-encoded proteins with DDX10 and their effect on DDX10 expression were further analyzed,and it was found that only the small vesicle membrane protein（E protein）could interact with DDX10 and significantly degrade the expression of DDX10 in a dose-dependent manner.In addition,expression of E protein alone could up-regulate the autophagy marker LC3-II,while NH₄Cl treatment and knockout of key autophagy genes ATG5/ATG7 block the degradation of DDX10 by E protein,suggesting that E protein degrades DDX10 through the autophagy lysosomal pathway.The nucleoplasm separation assay also further confirmed that E protein could induce the migration of DDX10 from the nucleus to the cytoplasm,while treatment with the autophagy inducer Rapamycin significantly reduced the amount of DDX10 in the cytoplasm but did not significantly change DDX10 amount in the nucleus,indicating that the degradation of DDX10 by E protein occurred in the cytoplasm.Co-IP assay was performed for screening the autophagy cargo receptors interacting with DDX10 or E protein,and it was found that only p62 interacted with DDX10 and promoted the degradation of DDX10 by E protein.The effect of knockout p62 on DDX10 expression was further analyzed,and the E protein was found to degrade DDX10 by selective autophagy in a p62-dependent manner.The findings suggest that DDX10 can inhibit PRRSV proliferation by activating IFN production,while the PRRSV-encoded E protein induces nucleoplasm migration of DDX10 in order to antagonize the antiviral effect of DDX10,and degrades DDX10 via the autophagy lysosomal pathway,thus helping PRRSV to achieve immune escape.