应用CRISPR/Cas9敲除HeLa细胞DDX21基因对新城疫病毒复制的影响

doi:10.11843/j.issn.0366-6964.2019.07.012

Abstract

Abstract: The helicase family is a class of enzymes that are essential to all living organisms. The main function of the helicase family is gene unwapping. The DEAD/H-box family RNA helicase is a kind of important helicase. DEAD-box RNA helicase DDX21 has been proved that it may regulate innate immunity of host cells. Newcastle disease virus (NDV) is a kind of pathogen which is harmful to poultry industry. NDV can regulate the innate immunity of the host by various means. In order to verify how DDX21 regulates NDV replication, this study used CRISPR/Cas9 system to establish a DDX21 knockout HeLa cell line, and to verify its effect on NDV infection. Six sgRNAs were designed to construct knockout vectors for DDX21 gene and electrotransfected into HeLa cells. After drug screening and subcloning, the knockout efficiency was identified by PCR and Western blot, and the replication efficiency of NDV in wild type and DDX21 knockout cells was compared. The results showed that only one DDX21 heterozygote knockout cell line was obtained because DDX21 is critical for cell growth. Western blot results showed that the expression of DDX21 was significantly inhibited. The infection ability of NDV to knockout cells was significantly lower than that of wild type cells, indicating that DDX21 played a positive role in regulating NDV replication. The research above laid a foundation for the study of DDX21 regulating antiviral innate immunity.

CLC Number:

S852.659.5

WU Wei, WANG Sa, MENG Chunchun, QIU Xusheng, LIAO Ying, TAN Lei, SONG Cuiping, LIU Weiwei, SUN Yingjie, DING Chan. Role of DDX21 Gene in Newcastle Disease Virus Replication by Using CRISPR/Cas9-Mediated Gene Knockout Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(7): 1433-1440.

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References

[1] WU Y. Unwinding and rewinding:double faces of helicase?[J]. J Nucleic Acids, 2012, 2012:140601.
[2] UMATE P, TUTEJA N, TUTEJA R. Genome-wide comprehensive analysis of human helicases[J]. Commun Integr Biol, 2011, 4(1):118-137.
[3] TANNER N K, LINDER P. DExD/H box RNA helicases:from generic motors to specific dissociation functions[J]. Mol Cell, 2001, 8(2):251-262.
[4] ABDELHALEEM M, MALTAIS L, WAIN H. The human DDX and DHX gene families of putative RNA helicases[J]. Genomics, 2003, 81(6):618-622.
[5] YONEYAMA M, KIKUCHI M, NATSUKAWA T, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses[J]. Nat Immunol, 2004, 5(7):730-737.
[6] SAITO T, HIRAI R, LOO Y M, et al. Regulation of innate antiviral defenses through a shared repressor domain in RIG-I and LGP2[J]. Proc Natl Acad Sci U S A, 2007, 104(2):582-587.
[7] YONEYAMA M, KIKUCHI M, MATSUMOTO K, et al. Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity[J]. J Immunol, 2005, 175(5):2851-2858.
[8] VALIENTE-ECHEVERRÍA F, HERMOSO M A, SOTO-RIFO R. RNA helicase DDX3:at the crossroad of viral replication and antiviral immunity[J]. Rev Med Virol, 2015, 25(5):286-299.
[9] ZHAO S C, GE X N, WANG X L, et al. The DEAD-box RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9in vitro[J]. Virus Res, 2015, 195:217-224.
[10] DONG Y C, YE W, YANG J, et al. DDX21 translocates from nucleus to cytoplasm and stimulates the innate immune response due to dengue virus infection[J]. Biochem Biophys Res Commun, 2016, 473(2):648-653.
[11] FLORES-ROZAS H, HURWITZ J. Characterization of a new RNA helicase from nuclear extracts of HeLa cells which translocates in the 5' to 3' direction[J]. J Biol Chem, 1993, 268(28):21372-21383.
[12] ZHANG Z Q, KIM T, BAO M S, et al. DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells[J]. Immunity, 2011, 34(6):866-878.
[13] CHEN G F, LIU C H, ZHOU L G, et al. Cellular DDX21 RNA helicase inhibits influenza A virus replication but is counteracted by the viral NS1 protein[J]. Cell Host Microbe, 2014, 15(4):484-493.
[14] ALEXANDER D J. Newcastle disease and other avian paramyxoviruses[J]. Rev Sci Tech, 2000, 19(2):443-462.
[15] FIOLA C, PEETERS B, FOURNIER P, et al. Tumor selective replication of Newcastle disease virus:association with defects of tumor cells in antiviral defence[J]. Int J Cancer, 2006, 119(2):328-338.
[16] HSU P D, LANDER E S, ZHANG F. Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014, 157(6):1262-1278.
[17] YU Z S, REN M D, WANG Z X, et al. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila[J]. Genetics, 2013, 195(1):289-291.
[18] HODROJ D, SERHAL K, MAIORANO D. Ddx19 links mRNA nuclear export with progression of transcription and replication and suppresses genomic instability upon DNA damage in proliferating cells[J]. Nucleus, 2017, 8(5):489-495.
[19] SONG C L, HOTZ-WAGENBLATT A, VOIT R, et al. SIRT7 and the DEAD-box helicase DDX21 cooperate to resolve genomic R loops and safeguard genome stability[J]. Genes Dev, 2017, 31(13):1370-1381.
[20] CALO E, FLYNN R A, MARTIN L, et al. RNA helicase DDX21 coordinates transcription and ribosomal RNA processing[J]. Nature, 2015, 518(7538):249-253.
[21] MA Z, MOORE R, XU X X, et al. DDX24 negatively regulates cytosolic RNA-mediated innate immune signaling[J]. PLoS Pathog, 2013, 9(10):e1003721.
[22] JANKNECHT R. Multi-talented DEAD-box proteins and potential tumor promoters:p68 RNA helicase (DDX5) and its paralog, p72 RNA helicase (DDX17)[J]. Am J Transl Res, 2010, 2(3):223-234.
[23] QIU X S, FU Q, MENG C C, et al. Newcastle disease virus V protein targets phosphorylated STAT1 to block IFN-I signaling[J]. PLoS One, 2016, 11(2):e0148560.
[24] SUN Y J, DONG L N, YU S Q, et al. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation[J]. FASEB J, 2017, 31(4):1337-1353.
[25] SUN Y J, YU S Q, DING N, et al. Autophagy benefits the replication of Newcastle disease virus in chicken cells and tissues[J]. J Virol, 2014, 88(1):525-537.

Role of DDX21 Gene in Newcastle Disease Virus Replication by Using CRISPR/Cas9-Mediated Gene Knockout Cells

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