Regulatory Role of DEAD-box Helicase 21 on Transmissible gastroenteritis virus Replication
To investigate the regulatory effect of DEAD-box helicase 21(DDX21)on the replication of Trans-missible gastroenteritis virus(TGEV).Firstly,Western Blot was utilized to analyze the effect of TGEV infection on DDX21 expression.Furthermore,we constructed eukaryotic expression plasmids of porcine DDX21 and established knockdown stable cell lines.RT-qPCR,Western Blot,Indirect immunofluorescence(IFA)and TCID50 assays were used to investigate the regulatory effect of DDX21 on TGEV replication in vitro.Western Blot analysis showed that the protein levels of DDX21 were significantly up-regulated in PK-15 cells at the early stage of TGEV infection.RT-qPCR,Western Blot,IFA and TCID50 experiments showed that over-expression of DDX21 significantly increased the mRNA level and protein of TGEV N in a dose-dependent manner.And the amino acids 601-784 aa of DDX21 were critical for promoting TGEV replication.Otherwise,the titer of TGEV was significantly down-regulated in DDX21 knockdown cell lines,whereas the titer of TGEV in DDX21 knockdown cell lines was reversed under the rescue ex-periment.This study revealed for the first time that DDX21 promotes the proliferation of TGEV and identified the key domain of DDX21 in regulating TGEV replication,which provided a theoretical a basis for future research on the function of DDX21 protein and the pathogenesis of TGEV.
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