| 41 | 0 | 173 |
| 下载次数 | 被引频次 | 阅读次数 |
目的 埃可病毒30型（Echovirus30, E30）作为肠道病毒B家族的重要成员，是引起无菌性脑膜炎、脑炎的主要病原体之一；目前在亚洲、欧洲以及南美洲呈季节性和周期性流行。然而，针对E30参与宿主感染的相关机制知之甚少。规律成簇间隔短回文重复/CRISPR相关核酸酶9(Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9, CRISPR/Cas9)系统已经被广泛用于对宿主基因的编辑，从而研究基因的生物学功能。方法本研究通过前期全基因筛选得到的宿主因子并运用CRISPR/Cas9技术针对构建跨膜蛋白240(Transmembrane protein 240, TMEM240)的外显子胞内环区域进行敲除，通过Western blot验证TMEM240蛋白完全敲除后得到单克隆敲除的人横纹肌肉瘤（Human rhabdomyosarcoma, RD）细胞，并探究TMEM240敲除后对E30复制的影响。结果 TMEM240的敲除能够显著抑制E30早期阶段的复制，但并不影响结合和内化阶段的复制。免疫荧光染色显示，TMEM240敲除后E30 dsRNA与早期核内体标志物Rab5A共定位显著减少，提示其参与E30早期核内体复制；转录组学分析表明，TMEM240相关差异基因富集于自噬体成熟、自噬体-溶酶体融合等通路。随后通过mCherry-GFP-LC3B融合腺病毒感染细胞试验、Western blot试验、共聚焦实验、溶酶体pH检测以及转录因子EB(Transcription factor EB, TFEB)核易位试验发现TMEM240的敲除/低能够影响自噬体-溶酶体的融合从而导致自噬体的累积以及TFEB的核转移。结论 本研究的结果显示TMEM240促进E30的复制，并且在自噬体-溶酶体的融合过程中发挥作用，这一发现为进一步了解E30的感染机制奠定基础。
Abstract：Objective Echovirus 30(E30), a prominent member of the Enterovirus B species, is one of the major causative agents of aseptic meningitis and encephalitis and exhibits seasonal and cyclical outbreaks in Asia, Europe and South America. However, the host factors and molecular mechanisms governing E30 infection remain poorly understood. The CRISPR/Cas9 genome-editing system provides a powerful approach for dissecting host gene function during viral infection. This study aimed to identify and characterize host factors essential for E30 replication. Methods Based on host factors identified in a prior genome-wide screen, CRISPR/Cas9-mediated gene editing was used to generate a knockout targeting the intracellular loop-encoding exon of transmembrane protein 240(TMEM240). Complete ablation of TMEM240 protein expression was confirmed by Western blotting in clonal human rhabdomyosarcoma(RD) cells. The impact of TMEM240 deficiency on E30 infection and replication was subsequently investigated. Results Knockout of TMEM240 significantly inhibited E30 replication at the early stage of infection, while viral binding and internalization were not affected. Immunofluorescence analysis demonstrated markedly reduced co-localization of E30 doublestranded RNA with the early endosome marker Rab5A in TMEM240-deficient cells, indicating a role for TMEM240 in early endosomal replication. Transcriptomic analysis revealed that TMEM240-associated differentially expressed genes were enriched in pathways related to autophagosome maturation and autophagosome–lysosome fusion. Furthermore, mCherry–GFP–LC3B adenoviral reporter assays, Western blotting, confocal microscopy, lysosomal pH measurements, and transcription factor EB(TFEB) nuclear translocation assays showed that loss or reduction of TMEM240 impaired autophagosome – lysosome fusion, leading to autophagosome accumulation and enhanced nuclear translocation of TFEB. Conclusion These findings identify TMEM240 as a proviral host factor that facilitates early E30 replication by regulating autophagosome–lysosome fusion. This study provides new mechanistic insight into E30–host interactions and advances our understanding of the molecular basis of E30 infection.
[1]Irvine DH, Irvine AB, Gardner PS. Outbreak of E. C. H. O. virus type 30 in a general practice[J]. Br Med J,1967, 4(5582):774-776. DOI:10. 1136/bmj. 4. 5582. 774.
[2]Bailly JL, Béguet A, Chambon M, et al. Nosocomial transmission of echovirus 30:molecular evidence by phylogenetic analysis of the VP1 encoding sequence[J].J Clin Microbiol, 2000, 38(8):2889-2892. DOI:10. 1128/JCM. 38. 8. 2889-2892. 2000.
[3]Rhoades RE, Tabor-Godwin JM, Tsueng G, et al.Enterovirus infections of the central nervous system[J].Virology, 2011, 411(2):288-305. DOI:10. 1016/j.virol. 2010. 12. 014.
[4]Duncan IB. An outbreak of aseptic meningitis associated with a previously unrecognized virus[J]. J Hyg, 1961,59(2):181-189. DOI:10. 1017/s0022172400038845.
[5]Gravelle CR, Noble GR, Feltz ET, et al. An epidemic of echovirus type 30 meningitis in an Arctic community[J]. Am J Epidemiol, 1974, 99(5):368-374. DOI:10. 1093/oxfordjournals. aje. a121623.
[6]Holmes CW, Koo SSF, Osman H, et al.Predominance of enterovirus B and echovirus 30 as cause of viral meningitis in a UK population[J]. J Clin Virol,2016, 81:90-93. DOI:10. 1016/j. jcv. 2016. 06. 007.
[7]Delplanque J, Devos D, Huin V, et al. TMEM240mutations cause spinocerebellar Ataxia 21 with mental retardation and severe cognitive impairment[J]. Brain,2014, 137(Pt 10):2657-2663. DOI:10. 1093/brain/awu202.
[8]Seki T, Sato M, Kibe Y, et al. Lysosomal dysfunction and early glial activation are involved in the pathogenesis of spinocerebellar Ataxia type 21 caused by mutant transmembrane protein 240[J]. Neurobiol Dis, 2018,120:34-50. DOI:10. 1016/j. nbd. 2018. 08. 022.
[9]Hu Q, Wang G, Chen X, et al. Neural-specific distribution of transmembrane protein TMEM240 and formation of TMEM240-Body[J]. Int J Biol Macromol, 2020, 161:692-703. DOI:10. 1016/j.ijbiomac. 2020. 06. 080.
[10]Jassey A, Wagner MA, Galitska G, et al. Starvation after infection restricts enterovirus D68 replication[J].Autophagy, 2023, 19(1):112-125. DOI:10. 1080/15548627. 2022. 2062888.
[11]Zhao X, Zhang G, Liu S, et al. Human neonatal fc receptor is the cellular uncoating receptor for enterovirus B[J]. Cell, 2019, 177(6):1553-1565. e16. DOI:10. 1016/j. cell. 2019. 04. 035.
[12]Kawai A, Uchiyama H, Takano S, et al.Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells[J]. Autophagy,2007, 3(2):154-157. DOI:10. 4161/auto. 3634.
[13]McWilliam Leitch EC, Bendig J, Cabrerizo M, et al.Transmission networks and population turnover of echovirus 30[J]. J Virol, 2009, 83(5):2109-2118.DOI:10. 1128/JVI. 02109-08.
[14]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. DOI:10. 13242/j.cnki. bingduxuebao. 004081.
[15]刘佳琪，李梦瑶，郝晨琳，等.利用CRISPR/Cas9技术构建RIPK1基因敲除的Neuro-2a细胞系及其功能研究[J].病毒学报，2025, 41(4):1121-1130. DOI:10. 13242/j. cnki. bingduxuebao. 240259.
[16]Diep J, Ooi YS, Wilkinson AW, et al. Enterovirus pathogenesis requires the host methyltransferase SETD3[J]. Nat Microbiol, 2019, 4(12):2523-2537. DOI:10. 1038/s41564-019-0551-1.
[17]Evans AS, Lennemann NJ, Coyne CB. BPIFB3interacts with ARFGAP1 and TMED9 to regulate noncanonical autophagy and RNA virus infection[J]. J Cell Sci, 2021, 134(5):jcs251835. DOI:10. 1242/jcs. 251835.
[18]Li Y, Jian X, Yin P, et al. Elucidating the host interactome of EV-A71 2C reveals viral dependency factors[J]. Front Microbiol, 2019, 10:636. DOI:10. 3389/fmicb. 2019. 00636.
[19]Baggen J, Thibaut HJ, Strating JRPM, et al. The life cycle of non-polio enteroviruses and how to target it[J].Nat Rev Microbiol, 2018, 16(6):368-381. DOI:10. 1038/s41579-018-0005-4.
[20]Shen D, Zhang G, Weng X, et al. A genome-wide CRISPR/Cas9 knockout screen identifies TMEM239 as an important host factor in facilitating African swine fever virus entry into early endosomes[J]. PLoS Pathog, 2024, 20(7):e1012256. DOI:10. 1371/journal. ppat. 1012256.
[21]Kuo CJ, Hansen M, Troemel E. Autophagy and innate immunity:Insights from invertebrate model organisms[J]. Autophagy, 2018, 14(2):233-242. DOI:10. 1080/15548627. 2017. 1389824.
[22]Ao X, Zou L, Wu Y. Regulation of autophagy by the rab GTPase network[J]. Cell Death Differ, 2014, 21(3):348-358. DOI:10. 1038/cdd. 2013. 187.
[23]Zou W, Wang X, Vale RD, et al. Autophagy genes promote apoptotic cell corpse clearance[J]. Autophagy,2012, 8(8):1267-1268. DOI:10. 4161/auto. 20786.
[24]Curnock R, Yalci K, Palmfeldt J, et al. TFEBdependent lysosome biogenesis is required for senescence[J]. EMBO J, 2023, 42(9):e111241. DOI:10. 15252/embj. 2022111241.
[25]Settembre C, Di Malta C, Polito VA, et al. TFEB links autophagy to lysosomal biogenesis[J]. Science,2011, 332(6036):1429-1433. DOI:10. 1126/science. 1204592.
[26]Fang S, Wan X, Zou X, et al. Arsenic trioxide induces macrophage autophagy and atheroprotection by regulating ROS-dependent TFEB nuclear translocation and AKT/mTOR pathway[J]. Cell Death Dis, 2021,12(1):88. DOI:10. 1038/s41419-020-03357-1.
[27]Ozcan M, Guo Z, Valenzuela Ripoll C, et al. Sustained alternate-day fasting potentiates doxorubicin cardiotoxicity[J]. Cell Metab, 2023, 35(6):928-942.e4. DOI:10. 1016/j. cmet. 2023. 02. 006.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250402
中图分类号:R373.24
引用信息:
[1]李冀琛,段伟,李慧洁,等.埃可病毒30型关键宿主因子TMEM240的鉴定及作用机制研究[J].病毒学报,2026,42(02):359-370.DOI:10.13242/j.cnki.bingduxuebao.250402.
基金信息:
国家疾病预防控制局公共卫生人才培养支持项目（项目号：GJJKJ-2024-ZY）; 传染病溯源预警与智能决策全国重点实验室（NITFID）资助项目（项目号：ZDGWNLJS25-36）,题目：未知及新发病原体监测及预测预警技术研究; 国家重点研发计划项目（项目号：2021YFC2302003）,题目：病毒监测网络数据标准及数据平台建设~~
2026-02-25
2026-02-25
2026-02-25