猪圆环病毒Ⅱ型感染3D4/2细胞的转录组学分析

doi:10.16431/j.cnki.1671-7236.2024.01.005

摘要/Abstract

摘要： 【目的】通过转录组测序筛选猪圆环病毒Ⅱ型（Porcine circovirus type Ⅱ，PCV2）感染猪肺泡巨噬细胞系（3D4/2）差异表达基因，为了解PCV2感染宿主免疫细胞机制和抗PCV2感染药物研发奠定基础。【方法】用感染复数（multiplicity of infection，MOI）为1的PCV2 NJ2002株处理3D4/2细胞，利用Illumina NovaSeq 6000测序平台进行转录组测序。使用DeSeq 2.0软件进行差异表达基因分析，并对差异表达基因进行基因本体论（GO）、京都基因与基因组百科全书（KEGG）功能分析及转录因子靶向分析，选取免疫及凋亡相关基因进行实时荧光定量PCR验证，用Western blotting技术检测细胞内磷脂酰肌醇激酶（PI3K）及磷酸化胞内磷脂酰肌醇激酶（p-PI3K）、蛋白激酶B （Akt）和磷酸化蛋白激酶B （p-Akt）蛋白表达水平。【结果】转录组测序结果显示，与对照组相比，PCV2感染组共获得713个差异表达基因，其中313个上调，400个下调。GO功能和KEGG通路富集分析显示，差异表达基因与免疫应答等相关，主要富集在细胞外基质受体互作通路、新陈代谢通路、HIF-1信号通路、病毒致癌作用、PI3K-Akt等信号通路。实时荧光定量PCR结果与转录组测序的基因表达水平保持一致。Western blotting检测结果显示，PCV2感染3D4/2细胞24 h后PI3K和Akt蛋白磷酸化水平显著升高（P<0.05）。转录因子靶向分析表明，差异表达基因与核转录因子Y亚基β（NFYB）和ETS转录因子（ELK4）联系紧密。【结论】PCV2可能通过PI3K-Akt信号通路影响下游炎症信号通路和凋亡信号通路增强自身复制能力。NFYB和ELK4转录因子在PCV2感染过程中可能发挥重要作用，可考虑作为抗PCV2药物靶点。研究结果为深入了解PCV2感染宿主免疫细胞机制和相关药物开发提供了理论基础。

关键词: 猪圆环病毒Ⅱ型; 3D4/2细胞; 转录组测序; 差异表达基因; 转录因子

Abstract: 【Objective】 The aim of this study was to screen the differentially expressed genes of Porcine circovirus type Ⅱ (PCV2) infected porcine alveolar macrophage cell line (3D4/2) by transcriptome sequencing, and lay a foundation for understanding the host immune cell mechanism of PCV2 infection and the development of anti-PCV2 infection drugs.【Method】 PCV2 NJ2002 strain with multiplicity of infection (MOI) equal to 1 was used to treat 3D4/2 cells, and transcriptome sequencing was performed using Illumina NovaSeq 6000 sequencing platform.DeSeq 2.0 software was used for differential expressed genes analysis.GO and KEGG function analysis and transcription factor targeting analysis were also performed for the differentially expressed genes.The genes related to immunity and apoptosis were selected for Real-time quantitative PCR verification, and the protein expression levels of intracellular phosphatidylinositol kinase (PI3K), phosphorylated intracellular phosphatidylinositol kinase (p-PI3K), protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt) were detected by Western blotting.【Result】 Transcriptome sequencing results showed that compared with the control group, 713 differentially expressed genes were obtained in PCV2 infection group, of which 313 were up-regulated and 400 were down-regulated. GO function and KEGG pathway enrichment analysis showed that differentially expressed genes were related to immune response, mainly concentrated in extracellular matrix receptor interaction pathway, metabolic pathway, HIF-1 signaling pathway, viral carcinogenesis, PI3K-Akt signaling pathway.The results of Real-time quantitative PCR were consistent with those of transcriptome sequencing.Western blotting results showed that the phosphorylation levels of PI3K and Akt were significantly increased after PCV2 infected 3D4/2 cells for 24 h (P<0.05).Transcription factor targeting analysis showed that differentially expressed genes were closely related to nuclear transcription factor Y subunit β (NFYB) and ETS transcription factor (ELK4).【Conclusion】 PCV2 might affect the downstream inflammatory signaling pathway and apoptosis signaling pathway to enhance its self-replication ability through the PI3K-Akt signaling pathway.NFYB and ELK4 transcription played important roles in PCV2 infection and could be considered as targets for anti-PCV2 drugs.The results provided theoretical basis for further understanding the mechanism of PCV2 infection in host immune cells and related drug development.

Key words: Porcine circovirus type Ⅱ; 3D4/2 cell; transcriptomics sequencing; differentially expressed genes; transcription factor

中图分类号:

S852.3

陈虹伶, 赵怡, 陈家骥, 韦秋旭, 李禧梦, 冯国越, 胡焜翔, 胡庭俊. 猪圆环病毒Ⅱ型感染3D4/2细胞的转录组学分析[J]. 中国畜牧兽医, 2024, 51(1): 42-51.

CHEN Hongling, ZHAO Yi, CHEN Jiaji, WEI Qiuxu, LI Ximeng, FENG Guoyue, HU Kunxiang, HU Tingjun. Transcriptomic Analysis of 3D4/2 Cells Infected with Porcine Circovirus Type Ⅱ[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(1): 42-51.

参考文献

[1] 孙庆帅, 陈燕虹, 姚伦, 等.猪圆环病毒2型和3型混合感染引起的母猪繁殖障碍诊治[J].中国畜牧兽医, 2022, 49(10):3982-3993. SUN Q S, CHEN Y H, YAO L, et al.Diagnosis treatment of reproductive disorders in sows caused by co-infection of PCV2 and PCV3[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(10):3982-3993.(in Chinese)
[2] MENG X J.Porcine circovirus type 2(PCV2):Pathogenesis and interaction with the immune system[J].Annual Review of Animal Biosciences, 2013, 1:43-64.
[3] FORT M, SIBILA M, NOFRARÍAS M, et al.Porcine circovirus type 2(PCV2) Cap and Rep proteins are involved in the development of cell-mediated immunity upon PCV2 infection[J].Veterinary Immunology and Immunopathology, 2010, 137(3-4):226-234.
[4] CORREA-FIZ F, FRANZO G, LLORENS A, et al.Porcine circovirus 2(PCV2) population study in experimentally infected pigs developing PCV2-systemic disease or a subclinical infection[J].Scientific Reports, 2020, 10(1):17747.
[5] DARWICH L, MATEU E.Immunology of Porcine circovirus type 2(PCV2)[J].Virus Research, 2012, 164(1-2):61-67.
[6] WEI R, VAN RENNE N, NAUWYNCK H J.Strain-dependent Porcine circovirus type 2(PCV2) entry and replication in T-lymphoblasts[J].Viruses, 2019, 11(9):813.
[7] WEI R, TRUS I, YANG B, et al.Breed differences in PCV2 uptake and disintegration in porcine monocytes[J].Viruses, 2018, 10(10):562.
[8] 李元曦, 夏应菊, 徐璐, 等.基于RNA-Seq对猪瘟病毒感染宿主的转录组学研究进展[J].中国兽药杂志, 2020, 54(11):79-85. LI Y X, XIA Y J, XU L, et al.Research progress of transcriptome of Classical swine fever virus after infection[J].Chinese Journal of Veterinary Drug, 2020, 54(11):79-85.(in Chinese)
[9] 李冀琛, 张国艳, 张珂艺, 等.基于RNA-Seq技术研究埃可病毒30型感染RD细胞前后的差异表达基因[J].病毒学报, 2022, 38(1):64-71. LI J C, ZHANG G Y, ZHANG K Y, et al.Exploration of differentially expressed genes before and after infection with Echovirus 30 based on RNA-sequencing[J].Chinese Journal of Virology, 2022, 38(1):64-71.(in Chinese)
[10] CHOI C Y, CHOI Y C, PARK I B, et al.The ORF5 protein of Porcine circovirus type 2 enhances viral replication by dampening type Ⅰ interferon expression in porcine epithelial cells[J].Veterinary Microbiology, 2018, 226:50-58.
[11] HE J, LENG C, PAN J, et al.Identification of lncRNAs involved in PCV2 infection of PK-15 cells[J].Pathogens, 2020, 9(6):479.
[12] LI C, SUN Y, LI J, et al.PCV2 regulates cellular inflammatory responses through dysregulating cellular miRNA-mRNA networks[J].Viruses, 2019, 11(11):1055.
[13] TANG S Y, WAN Y P, WU Y M.Death domain associated protein (Daxx), a multi-functional protein[J].Cellular & Molecular Biology Letters, 2015, 20(5):788-797.
[14] MAILLET S, NISOLE S.Daxx, a broad-spectrum viral restriction factor[J].Virologie, 2016, 20(5):261-272.
[15] SCHREINER S, WODRICH H.Virion factors that target Daxx to overcome intrinsic immunity[J].Journal of Virology, 2013, 87(19):10412-10422.
[16] WANG Y, SONG M, ZHOU P, et al.TNFAIP3-upregulated RIP3 exacerbates acute pancreatitis via activating NLRP3 inflammasome[J].International Immunopharmacology, 2021, 100:108067.
[17] ZHANG T, HU J, WANG X, et al.microRNA-378 promotes hepatic inflammation and fibrosis via modulation of the NF-κB-TNFα pathway[J].Journal of Hepatology, 2019, 70(1):87-96.
[18] NGUYEN L P, TRAN S C, SUETSUGU S, et al.PACSIN2 interacts with nonstructural protein 5A and regulates Hepatitis C virus assembly[J].Journal of Virology, 2020, 94(5):1531.
[19] VAN DUYNE R, FREED E O.HIV-1 packs in PACSIN2 for cell-to-cell spread[J].Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(27):6885-6887.
[20] MARTENS S, BRIDELANCE J, ROELANDT R, et al.MLKL in cancer:More than a necroptosis regulator[J].Cell Death and Differentiation, 2021, 28(6):1757-1772.
[21] PASPARAKIS M, VANDENABEELE P.Necroptosis and its role in inflammation[J].Nature, 2015, 517(7534):311-320.
[22] ZHANG T, YIN C, BOYD D F, et al.Influenza virus Z-RNAs induce ZBP1-mediated necroptosis[J].Cell, 2020, 180(6):1115-1129.
[23] YANG J, TAN H L, GU L Y, et al.Sophora subprosrate polysaccharide inhibited cytokine/chemokine secretion via suppression of histone acetylation modification and NF-κb activation in PCV2 infected swine alveolar macrophage[J]. International Journal of Biological Macromolecules, 2017, 104:900-908.
[24] XIAN H, WATARI K, SANCHEZ-LOPEZ E, et al.Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling[J].Immunity, 2022, 55(8):1370-1385.
[25] THARYAN R G, ANNIBAL A, SCHIFFER I, et al.NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin[J].Nature Metabolism, 2020, 2(5):387-396.
[26] JIANG X, NEVINS J R, SHATS I, et al.E2F1-mediated induction of NFYB attenuates apoptosis via joint regulation of a pro-survival transcriptional program[J]. PLoS One, 2015, 10(6):e0127951.
[27] MAURICE D, COSTELLO P, SARGENT M, et al.ERK signaling controls innate-like CD8⁺ T cell differentiation via the ELK4(SAP-1) and ELK1 transcription factors[J].Journal of Immunology, 2018, 201(6):1681-1691.
[28] ZHENG L, XU H, DI Y, et al.ELK4 promotes the development of gastric cancer by inducing M2 polarization of macrophages through regulation of the KDM5A-PJA2-KSR1 axis[J].Journal of Translational Medicine, 2021, 19(1):342.