SYNGR2影响猪圆环病毒2型体外增殖的研究

doi:10.11843/j.issn.0366-6964.2023.09.022

摘要/Abstract

摘要： 宿主的Synaptogyrin-2(SYNGR2)蛋白影响猪圆环病毒2型(porcine circovirus type 2,PCV2)的感染能力。本研究旨在研究宿主因子SYNGR2对PCV2在细胞水平增殖的影响,并进一步对SYNGR2影响PCV2体外增殖的机制进行探究。本研究利用基因敲除、过表达、定点突变等手段,在PK15细胞上研究SYNGR2基因敲除及其p.Arg63Cys点突变对PCV2增殖的影响。为探究SYNGR2参与PCV2感染的具体机制,作者对SYNGR2基因敲除的细胞及野生型对照细胞进行转录组测序,筛选SYNGR2功能相关的基因及通路,根据差异表达基因进行表达模式聚类分析,并通过实时荧光定量PCR(quantitative PCR,qPCR)验证差异表达基因的表达情况。结果显示,SYNGR2基因敲除或p.Arg63Cys点突变显著降低PCV2对PK15细胞的感染能力,对敲除细胞过表达不同基因型的SYNGR2,PCV2的增殖能力均恢复。转录组差异表达基因分析结果显示,SYNGR2的功能主要是参与膜成分及囊泡等被膜细胞器的组成。对差异表达基因肌球蛋白VIIA与Rab互作蛋白(myosin VIIA and Rab interacting protein,MYRIP)及其结合的外泌体调控相关基因Ras相关蛋白27(Ras-related protein-27,RAB27)进行qPCR检测,定量结果显示,RAB27 mRNA表达量在SYNGR2敲除后显著提升。本研究在细胞水平证实了SYNGR2参与PCV2的体外增殖,SYNGR2基因敲除显著降低PCV2在PK15上的增殖能力,同时SYNGR2第63位氨基酸位点由Arg突变为Cys也显著降低PCV2在PK15上的增殖能力。转录组分析揭示SYNGR2可能通过调节囊泡运输的功能来影响PCV2的复制能力。

关键词: SYNGR2, 猪圆环病毒2型, 转录组测序, RAB27A, 囊泡

Abstract: Synaptogyrin-2 (SYNGR2) protein affects PCV2 infection in host. This study aims to investigate the SYGNR2 function on PCV2 proliferation in cell cultures and dissect the mechanism for SYNGR2 affecting PCV2 infection. We performed gene knockout, overexpression and site-directed mutagenesis assay to study how SYNGR2 affects PCV replication in PK15 cells. To investigate the mechanism, RNA-seq was performed to study the molecular differences between SYNGR2 KO and WT cells. Differentially expressed genes between the two groups were analyzed and further validated by quantitative Real-time PCR. SYNGR2 knockout and p.Arg63Cys point mutation decreased PCV2 replication in PK15 cells, and the overexpression of different genotypes of SYNGR2 restored PCV2 replication capacity in SYNGR2 KO cells. RNA-seq enrichment analysis showed that the SYNGR2 mainly participates in the membrane components and vesicles and other membrane organelles. The differentially expressed gene MYRIP and its bound exosome regulation related gene RAB27A were selected for verification, and qPCR results showed that the expression of RAB27A mRNA was significantly increased in SYNGR2 knockout cells.In Conclusion, this report confirmed SYNGR2 function to support PCV2 infection in cells. SYNGR2 knockout and p.Arg63Cys point mutation both decreased PCV2 replication in cells. Transcriptome analysis reveals that SYNGR2 may affect PCV2 replication capacity by regulating the function of vesicular transport.

Key words: SYNGR2, PCV2, RNA-seq, RAB27A, vesicle

中图分类号:

S855.3

梁凯欣, 钟海文, 宋长绪, 杨化强, 黄思秀, 徐铮. SYNGR2影响猪圆环病毒2型体外增殖的研究[J]. 畜牧兽医学报, 2023, 54(9): 3824-3835.

LIANG Kaixin, ZHONG Haiwen, SONG Changxu, YANG Huaqiang, HUANG Sixiu, XU Zheng. Study on the Effect of SYNGR2 on PCV2 Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3824-3835.

参考文献

[1] XU P L, ZHAO Y, ZHENG H H, et al. Analysis of genetic variation of porcine circovirus type 2 within pig populations in central China[J]. Arch Virol, 2019, 164(5):1445-1451.
[2] WANG F, GUO X, GE X N, et al. Genetic variation analysis of Chinese strains of porcine circovirus type 2[J]. Virus Res, 2009, 145(1):151-156.
[3] ALLAN G M, MCNEILLY F, ELLIS J, et al. Experimental infection of colostrum deprived piglets with porcine circovirus 2(PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV2 replication[J]. Arch Virol, 2000, 145(11):2421-2429.
[4] DORR P M, BAKER R B, ALMOND G W, et al. Epidemiologic assessment of porcine circovirus type 2 coinfection with other pathogens in swine[J]. J Am Vet Med Assoc, 2007, 230(2):244-250.
[5] ELLIS J A, ALLAN G, KRAKOWKA S. Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnotobiotic pigs[J]. Am J Vet Res, 2008, 69(12):1608-1614.
[6] ALLAN G M, ELLIS J A. Porcine circoviruses:a review[J]. J Vet Diagn Invest, 2000, 12(1):3-14.
[7] YU S, OPRIESSNIG T, KITIKOON P, et al. Porcine circovirus type 2(PCV2) distribution and replication in tissues and immune cells in early infected pigs[J]. Vet Immunol Immunopathol, 2007, 115(3-4):261-272.
[8] CAO J J, LIN C, WANG H J, et al. Circovirus transport proceeds via direct interaction of the cytoplasmic dynein IC1 subunit with the viral capsid protein[J]. J Virol, 2015, 89(5):2777-2791.
[9] GUO K K, XU L, WU M M, et al. A host factor GPNMB restricts porcine circovirus type 2(PCV2) replication and interacts with PCV2 ORF5 protein[J]. Front Microbiol, 2019, 9:3295.
[10] SUN R J, SUN S Q, ZHANG Y K, et al. PCV2 induces reactive oxygen species to promote nucleocytoplasmic translocation of the viral DNA binding protein HMGB1 to enhance its replication[J]. J Virol, 2020, 94(13):e00238-20.
[11] STEVENS R J, AKBERGENOVA Y, JORQUERA R A, et al. Abnormal synaptic vesicle biogenesis in Drosophila synaptogyrin mutants[J]. J Neurosci, 2012, 32(50):18054-18067.
[12] KANDROR K V, PILCH P F. The sugar is sIRVed:sorting glut4 and its fellow travelers[J]. Traffic, 2011, 12(6):665-671.
[13] SUN Q Y, QI X, ZHANG Y, et al. Synaptogyrin-2 promotes replication of a novel tick-borne bunyavirus through interacting with viral nonstructural protein NSs[J]. J Biol Chem, 2016, 291(31):16138-16149.
[14] BOESZE-BATTAGLIA K, DHINGRA A, WALKER L M, et al. Internalization and intoxication of human macrophages by the active subunit of the Aggregatibacter actinomycetemcomitans cytolethal distending toxin is dependent upon cellugyrin (synaptogyrin-2)[J]. Front Immunol, 2020, 11:1262.
[15] WALKER L R, ENGLE T B, VU H, et al. Synaptogyrin-2 influences replication of porcine circovirus 2[J]. PLoS Genet, 2018, 14(10):e1007750.
[16] PILERI P, UEMATSU Y, CAMPAGNOLI S, et al. Binding of hepatitis C virus to CD81[J]. Science, 1998, 282(5390):938-941.
[17] EARNEST J T, HANTAK M P, LI K, et al. The tetraspanin CD9 facilitates MERS-coronavirus entry by scaffolding host cell receptors and proteases[J]. PLoS Pathog, 2017, 13(7):e1006546.
[18] DZIDUSZKO A, OZBUN M A. Annexin A2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in Human keratinocytes[J]. J Virol, 2013, 87(13):7502-7515.
[19] FU E Q, PAN L, XIE Y H, et al. Tetraspanin CD63 is a regulator of HIV-1 replication[J]. Int J Clin Exp Pathol, 2015, 8(2):1184-1198.
[20] KREMENTSOV D N, WENG J, LAMBELÉ M, et al. Tetraspanins regulate cell-to-cell transmission of HIV-1[J]. Retrovirology, 2009, 6:64.
[21] BELFORT G M, KANDROR K V. Cellugyrin and synaptogyrin facilitate targeting of synaptophysin to a ubiquitous synaptic vesicle-sized compartment in PC12 cells[J]. J Biol Chem, 2003, 278(48):47971-47978.
[22] OSTROWSKI M, CARMO N B, KRUMEICH S, et al. Rab27a and Rab27b control different steps of the exosome secretion pathway[J]. Nat Cell Biol, 2010, 12(1):19-30.
[23] 陈洪博, 段滇宁, 胡瑶, 等. 猪圆环病毒2型感染PK-15细胞中外泌体的鉴定及其对细胞增殖和凋亡的影响[J]. 畜牧兽医学报, 2020, 51(7):1688-1698.
CHEN H B, DUAN D N, HU Y, et al. Identification of exosomes in porcine circovirus type 2 infected PK-15 cells and its effects on cell proliferation and apoptosis[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7):1688-1698. (in Chinese)
[24] XU G W, XU S X, SHI X J, et al. Intercellular transmission of Seneca Valley virus mediated by exosomes[J]. Vet Res, 2020, 51(1):91.
[25] WANG T, FANG L R, ZHAO F W, et al. Exosomes mediate intercellular transmission of porcine reproductive and respiratory syndrome virus[J]. J Virol, 2018, 92(4):e01734-17.
[26] LONGATTI A. The dual role of exosomes in hepatitis A and C virus transmission and viral immune activation[J]. Viruses, 2015, 7(12):6707-6715.
[27] WALKER L R. Functional and evolutionary analysis of host synaptogyrin-2 in porcine circovirus type 2 susceptibility[D]. Lincoln:The University of Nebraska, 2022.
[28] BELLO-MORALES R, CRESPILLO A J, FRAILE-RAMOS A, et al. Role of the small GTPase Rab27a during Herpes simplex virus infection of oligodendrocytic cells[J]. BMC Microbiol, 2012, 12:265.