Regulatory Role of DEAD-box Helicase 21 on Transmissible gastroenteritis virus Replication

doi:10.7668/hbnxb.20194747

Abstract

Abstract:

To investigate the regulatory effect of DEAD-box helicase 21(DDX21)on the replication of Transmissible 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 experiment.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.

Key words: Transmissible gastroenteritis virus, DEAD-box helicase 21, Host protein, Viral replication

XIE Lilan, YIN Jie, HAUNG Donge, LI Yaoming. Regulatory Role of DEAD-box Helicase 21 on Transmissible gastroenteritis virus Replication[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 215-222. doi: 10.7668/hbnxb.20194747.

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Figures/Tables 7

Tab.1 The PCR primers used in this study

引物名称Primer name	引物序列Primer sequence	备注Notes
GAPDH-F	ACATGGCTCCAAGGAGTAAGA	RT-qPCR
GAPDH-R	GATCGAGTTGGGGCTGTGACT
JC-TGEV-N-F	CCCGTGGTCGGAAGAATAATAA	RT-qPCR
JC-TGEV-N-R	GGTACAAAGTCTCTCGGACATAAG
DDX21-F	GGCAAGTCAAGTAAGCAGAGA	RT-qPCR
DDX21-R	GCGATCAATCTGTCCTCCATAG
DDX21-FF	GGAGAATTCATGCCGGGGAAACTTCGTAGT	构建重组质粒HA-DDX21wt及截短
DDX21-FR	TCTCTCGAGTTACTGTCCAAACGCTTTGC	突变体质粒ΔGUCT、DEAD和GUCT
DDX21-600R	TCTCTCGAGTTACTCAGCTGACTGCTTGAAG
DDX21-430R	TATCTCGAGTTAATCCCCGATAACTGCTGCC
DDX21-601F	GGAGAATTCATGAAGCTGATAGAGGAGAAGG
PL548F	CAAACTTAAGCATGTTGT	构建回补突变体质粒HA-DDX21m
PL548R	TCTCAGCTGACTGCTTGA
PL548F1	ATGTGTCGCCTATAAAAAAGATTCAGAAGAC
PL548R1	TTATAGGCGACACATAAAATCTCT

Fig.1 TGEV infection regulates the expression of endogenous DDX21 A.The change of DDX21 protein expression levels in TGEV-infected cells(+means virus-infected,-means uninfected);B.Gray values were analyzed by Image J soft(+/-).The difference in small letters between different groups at the same time is significant(P<0.05).

Fig.2 Construction of eukaryotic expression plasmid HA-DDX21wt and detection of its expression by Western Blot A.Identification of recombinant plasmid HA-DDX21wt by restriction enzyme digestion:M1.DL2000 Marker;M2.DL15000 Marker;B.Western Blot analysis of recombinant plasmid expression.

Fig.3 Over expression of DDX21 positively regulates the replication of TGEV A—C.TCID₅₀(A),Real-time Fluorescence Quantification(B)and Western Blot(C)were used to detect the effect of DDX21 on TGEV proliferation;D.DDX21 regulates TGEV proliferation in a dose-dependent manner;different small letters indicate the significant difference at P<0.05.The same as Fig.4—6.

Fig.4 Knockdown of DDX21 expression inhibits replication of TGEV A.The expressions of DDX21 were measured by Western Blot; B—C.The replication of TGEV were detected by TCID₅₀ assay(B)and qRT-PCR assay(C),respectively.

Fig.5 Construction and functional analysis of the rescue plasmid A.Mutation bases;B.Sequencing map(partial)of the complement plasmid;C.Rescue experiment.

Fig.6 Effect of different DDX21 domains on TGEV proliferation A.Schematic showing the DDX21 deletion mutants;B.TCID₅₀ assay was used to detect the effects of DDX21 domains on TGEV proliferation.

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