MLKL基因敲除对猪伪狂犬病病毒复制的影响

doi:10.16431/j.cnki.1671-7236.2022.05.034

Abstract

Abstract: 【Objective】 This study was aimed to construct knockout mixed lineage kinase domain-like (MLKL) PK-15 cell line,and investigate the effects of MLKL gene knockout on the replication of Pseudorabies virus (PRV).【Method】 The MLKL-sgRNA editing vector was constructed by designing specific editing sites based on MLKL sequences using CRISPR/Cas9 gene editing technology,and transfected into PK-15 cell.The polyclonal cell line was obtained by puromycin screening,and PK-15 MLKL-KO monoclonal cell line was obtained by limited dilution method.The knockout level of MLKL gene on PK-15 cell was verified by target genomic PCR,sequencing and Western blotting.Viral proliferation levels were detected using Reed-Muench method.PI staining and fluorescence microscopy were used to observe cell necrosis.【Result】 The results showed that one PK-15 cell line with a 647 bp deletion of MLKL gene was screened,and the expression of MLKL protein was not detected by Western blotting.Compared with PK-15 cell,PK-15 MLKL-KO cell extremely significantly or significantly increased the viral titers of PRV GD-WH (expect for 36 h post infection) and PRV Bartha-K61 (P<0.01 or P<0.05).After PRV GD-WH and PRV Bartha-K61 were infected with PK-15 MLKL-KO cells,the necrotic cells were significantly reduced.【Conclusion】 The MLKL gene knockout PK-15 cell line was constructed in this study.Compared with PK-15 cell,PK-15 MLKL-KO cell significantly improved the replication and survival of PRV GD-WH and PRV Bartha-K61,providing a feasible strategy to increase virus yield during PRV Bartha-K61 vaccine production.

Key words: MLKL gene; CRISPR/Cas9 gene editing technology; knockout; PK-15 cell; Pseudorabies virus (PRV)

CLC Number:

S852.65⁺9.1

XIE Sihao, GOU Hongchao, BIAN Zhibiao, LI Bin, CAI Rujian, ZANG Yingan, LI Chunling. Effects of MLKL Gene Knockout on Replication of Pseudorabies Virus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1934-1941.

References

[1] PUSCHNIK A S,MAJZOUB K,OOI Y S,et al.A CRISPR toolbox to study virus-host interactions[J].Nature Reviews Microbiology,2017,15(6):351-364.
[2] PATRICK D H,ERIC S L,FENG Z.Development and applications of CRISPR-Cas9 for genome engineering[J].Cell,2014,157(6):1262-1278.
[3] 景润春,卢洪.CRISPR/Cas9基因组定向编辑技术的发展与在作物遗传育种中的应用[J].中国农业科学,2016,49(7):1219-1229. JING R C,LU H.The development of CRISPR/Cas9 system and its application in crop genome editing[J].Scientia Agricultura Sinica,2016,49(7):1219-1229.(in Chinese)
[4] 张爽,樊爽爽,常雯茹,等.干扰素调节因子3基因敲除对伪狂犬病病毒增殖的影响[J].中国畜牧兽医,2019,46(5):1253-1262. ZHANG S,FAN S S,CHANG W R,et al.Effect of interferon regulatory factor 3 gene knockout on proliferation of Pseudorabies virus[J].China Animal Husbandry & Veterinary Medicine,2019,46(5):1253-1262.(in Chinese)
[5] 张越秀,张华伟,李连峰,等.利用CRISPR/Cas9技术构建OAS2敲除的PK-15细胞系及敲除OAS2对CSFV复制的影响[J].中国预防兽医学报,2019,41(11):1094-1098. ZHANG Y X,ZHANG H W,LI L F,et al.Construction of OAS2 knockout PK-15 cell line and its effect on the replication of Classical swine fever virus[J].Chinese Journal of Preventive Veterinary Medicine,2019,41(11):1094-1098.(in Chinese)
[6] 谷思睿,赵军,陈弟诗,等.伪狂犬病毒潜伏感染相关病毒因子研究进展[J].病毒学报,2021,37(6):1-10. GU S R,ZHAO J,CHEN D S,et al.Research progress in viral factors associated with latent infection of Pseudorabies virus[J].Chinese Journal of Virology,2021,37(6):1-10.(in Chinese)
[7] SARAH D C,GREGORY A C,BRADLEY L N,et al.Pseudorabies virus infection in Oklahoma hunting dogs[J].Journal of Veterinary Diagnostic Investigation,2011,23(5):915-923.
[8] 常雯茹,段利芳,杨乐,等.伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究[J].中国畜牧兽医,2021,48(1):83-92. CHANG W R,DUAN L G,YANG L,et al.Study on replication patterns of Pseudorabies virus in cell lines with NF-κB family p65 gene knocked out[J].China Animal Husbandry & Veterinary Medicine,2021,48(1):83-92.(in Chinese)
[9] LI H,LIANG R,PANG Y,et al.Evidence for interspecies transmission route of Pseudorabies virus via virally contaminated fomites[J].Veterinary Microbiology,2020,251:108912.
[10] RZIHA H,METTENLEITER T,OHLINGER V,et al.Herpesvirus Pseudorabies virus latency in swine:Occurrence and physical state of viral DNA in neural tissues[J].Virology,1986,155(2):600-613.
[11] ZHOU J Z,LI S,WANG X B,et al.Bartha-K61 vaccine protects growing pigs against challenge with an emerging variant Pseudorabies virus[J].Vaccine,2017,35(8):1161-1166.
[12] WANG H Y,SUN L M,SU L J,et al.Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3[J].Molecular Cell,2014,54(1):133-146.
[13] SUN L M,WANG H Y,WANG Z G,et al.Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase[J].Cell,2012,148(1-2):213-227.
[14] CAI Z Y,JITKAEW S,ZHAO J,et al.Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis[J].Nature Cell Biology,2014,16(2):55-65.
[15] HE S D,WANG L,MIAO L,et al.Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-α[J].Cell,2009,137(6):1100-1111.
[16] ZHAO J,JITKAEW S,CAI Z Y,et al.Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(14):5322-5327.
[17] CHEN X,LI W J,REN J M,et al.Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death[J].Cell Research,2014,24(1):105-121.
[18] GOU H C,BIAN Z B,CAI R J,et al.RIPK3-dependent necroptosislimits PRV replication in PK-15 cells[J].Frontiers in Microbiology,2021,12:664353.
[19] CONG L,RAN F A,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[20] SUI C,JIANG D D,WU X J,et al.CRISPR-Cas9 mediated RNase L knockout regulates cellular function of PK-15 cells and increases PRV replication[J].BioMed Research International,2019,2019:398208.
[21] WU W,ORR-BURKS N,KARPILOW J,et al.Development of improved vaccine cell lines against Rotavirus[J].Scientific Data,2017,4:170021.
[22] HOEKSEMA F,KARPILOW J,LUITJENS A,et al.Enhancing viral vaccine production using engineered knockout Vero cell lines——A second look[J].Vaccine,2018,36(16):2093-2103.
[23] ORR-BURKS N,MURRAY J,WU W,et al.Gene-edited Vero cells as Rotavirus vaccine substrates[J].Vaccine:X,2019,3:100045.
[24] WANG W,WANG W H,AZADZOI K M,et al.Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis[J].Scientific Reports,2016,6:22550.
[25] WANG L L,WANG T T,LI H B,et al.Receptor interacting protein 3-mediated necroptosis promotes lipopolysaccharide-induced inflammation and acute respiratory distress syndrome in mice[J].PLoS One,2016,11(5):e0155723.
[26] HARRIS K G,MOROSKY S A,DRUMMOND C G,et al.RIP3 regulates autophagy and promotes Coxsackievirus B3 infection of intestinal epithelial cells[J].Cell Host & Microbe,2015,18(2):221-232.
[27] WANG X,LI Y,LIU S,et al.Direct activation of RIP3/MLKL-dependent necrosis by Herpes simplex virus 1 (HSV-1) protein ICP6 triggers host antiviral defense[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(43):15438-15443.
[28] CHO Y S,CHALLA S,MOQUIN D,et al.Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation[J].Cell,2009,137(6):1112-1123.
[29] WEN C X,YU Y F,GAO C F,et al.RIPK3-dependent necroptosisis induced and restricts viral replication in human astrocytes infected with Zikavirus[J].Frontiers in Cellular and Infection Microbiology,2021,11:637710.
[30] NOGUSA S,THAPA R J,DILLON C P,et al.RIPK3 activates parallel pathways of MLKL-driven necroptosis and FADD-mediated apoptosis to protect against Influenza A virus[J].Cell Host & Microbe,2016,20(1):13-24.

Effects of MLKL Gene Knockout on Replication of Pseudorabies Virus

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