| 19 | 0 | 229 |
| 下载次数 | 被引频次 | 阅读次数 |
目的 探究巨病毒中是否存在单链DNA结合蛋白（Single-stranded DNA-binding protein, SSB），并通过实验验证其功能。方法 利用原核表达系统表达并纯化Crov372蛋白，纯化的蛋白质采用Western blot验证并通过电泳迁移率实验检测其与单链DNA的结合能力；采用AlphaFold2对Crov372蛋白质进行三级结构预测，根据预测结果对该蛋白质进行截短、取代突变和缺失突变，研究Crov372蛋白与核酸结合调控的关键区域。结果 表达纯化得到Crov372蛋白，三级结构预测显示，该蛋白主要由N端的寡核苷酸结合折叠和长约100个酸性氨基酸富集的C端无序尾巴组成。电泳迁移率实验结果显示全长Crov372蛋白与单链DNA结合能力较弱；对C端进行截短后，截短体随着C端长度的变短，与核酸结合能力显著增强。对C端无序区的一段酸性氨基酸聚集区进行取代突变与缺失突变实验，结果表明减少酸性残基在C端的比例可增强结合能力，该酸性氨基酸簇在调控单链DNA结合蛋白与单链DNA结合中起关键作用。结论 本研究通过实验证实巨病毒中Crov372的单链DNA结合蛋白功能，并揭示其C端无序区对结合活性具有负调控作用，其中酸性氨基酸簇是调控与单链DNA结合的关键区域，为理解SSB蛋白在巨病毒复制中的作用提供了新线索。
Abstract：Objective To investigate whether giant viruses encode single-stranded DNA-binding(SSB) proteins and to experimentally characterize the function of the candidate protein Crov372. Method Crov372 was expressed and purified using a prokaryotic expression system. Protein expression and purity were verified by Western blotting, and its binding affinity for single-stranded DNA(ssDNA) was assessed using electrophoretic mobility shift assays(EMSA). The tertiary structure of Crov372 was predicted using AlphaFold2. Based on the structural model, truncation, substitution, and deletion mutants were generated to identify regions critical for nucleic acid binding. Results Recombinant Crov372 was successfully expressed and purified. Structural prediction indicated that Crov372 comprises an N-terminal oligonucleotide/oligosaccharide-binding(OB) fold and a C-terminal intrinsically disordered region enriched in approximately 100 acidic residues. EMSA demonstrated that full-length Crov372 exhibits relatively weak ssDNA-binding activity. Progressive truncation of the C-terminal tail markedly enhanced ssDNA-binding affinity. Substitution and deletion analyses targeting an acidic residue cluster within the C-terminal disordered region showed that reducing the proportion of acidic residues significantly increased binding affinity, indicating that this cluster plays a key regulatory role in modulating ssDNA binding. Conclusion This study demonstrates that Crov372 functions as a single-stranded DNA-binding protein in giant viruses and reveals that its C-terminal intrinsically disordered region negatively regulates DNA-binding activity. The acidic residue cluster within this region is critical for controlling ssDNA binding. These findings provide new insights into the role of SSB proteins in the replication of giant viruses.
[1]Antony E, Lohman TM. Dynamics of E. coli single stranded DNA binding(SSB)protein-DNA complexes[J]. Semin Cell Dev Biol, 2019, 86:102-111. DOI:10. 1016/j. semcdb. 2018. 03. 017.
[2]张珊珊，汪铭书，程安春，等.单纯疱疹病毒1型单链结合蛋白ICP8研究进展[J].病毒学报，2023, 39(4):1172-1180. DOI:10. 13242/j. cnki.bingduxuebao. 004351.
[3]Bianco PR. The mechanism of action of the SSB interactome reveals it is the first OB-fold family of genome guardians in prokaryotes[J]. Protein Sci,2021, 30(9):1757-1775. DOI:10. 1002/pro. 4140.
[4]Theobald DL, Mitton-Fry RM, Wuttke DS. Nucleic acid recognition by OB-fold proteins[J]. Annu Rev Biophys Biomol Struct, 2003, 32:115-133. DOI:10. 1146/annurev. biophys. 32. 110601. 142506.
[5]Byrne BM, Oakley GG. Replication protein A, the laxative that keeps DNA regular:The importance of RPA phosphorylation in maintaining genome stability[J]. Semin Cell Dev Biol, 2019, 86:112-120. DOI:10. 1016/j. semcdb. 2018. 04. 005.
[6]Marintcheva B, Hamdan SM, Lee SJ, et al. Essential residues in the C terminus of the bacteriophage T7 gene2. 5 single-stranded DNA-binding protein[J]. J Biol Chem, 2006, 281(35):25831-25840. DOI:10. 1074/jbc. M604601200.
[7]Yin J, Fu Y, Rao G, et al. Structural transitions during the cooperative assembly of baculovirus single-stranded DNA-binding protein on ssDNA[J]. Nucleic Acids Res, 2022, 50(22):13100-13113. DOI:10. 1093/nar/gkac1142.
[8]Nowak M, Olszewski M，Śpibida M, et al.Characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila, Flavobacterium psychrophilum,Psychrobacter arcticus, Psychrobacter cryohalolentis,Psychromonas ingrahamii, Psychroflexus torquis, and Photobacterium profundum[J]. BMC Microbiol, 2014,14(1):91. DOI:10. 1186/1471-2180-14-91.
[9]Arthofer P, Panhölzl F, Delafont V, et al. A giant virus infecting the amoeboflagellate Naegleria[J]. Nat Commun, 2024, 15:3307. DOI:10. 1038/s41467-024-47308-2.
[10]Colson P, La Scola B, Levasseur A, et al. Mimivirus:leading the way in the discovery of giant viruses of amoebae[J]. Nat Rev Microbiol, 2017, 15(4):243-254. DOI:10. 1038/nrmicro. 2016. 197.
[11]Mönttinen HAM, Bicep C, Williams TA, et al. The genomes of nucleocytoplasmic large DNA viruses:viral evolution writ large[J]. Microb Genom, 2021, 7(9).DOI:10. 1099/mgen. 0. 000649.
[12]Brandes N, Linial M. Giant viruses—big surprises[J].Viruses, 2019, 11(5):404. DOI:10. 3390/v11050404.
[13]龚超文，周雪雯，潘迎捷，等.噬病毒体启动子模体的初步预测分析[J].病毒学报，2015, 31(4):395-403.DOI:10. 13242/j. cnki. bingduxuebao. 002734.
[14]Fischer MG, Kelly I, Foster LJ, et al. The virion of Cafeteria roenbergensis virus(CroV)contains a complex suite of proteins for transcription and DNA repair[J]. Virology, 2014, 466:82-94. DOI:10. 1016/j. virol. 2014. 05. 029.
[15]Grimsley GR, Pace CN. Spectrophotometric determination of protein concentration[J]. Curr Protoc Protein Sci, 2003, 33(1):3. 1. 1-3. 1. 9. DOI:10. 1002/0471140864. ps0301s33.
[16]Tian FJ, Zhang C, Zhou E, et al. Universality in RNA and DNA deformations induced by salt, temperature change, stretching force, and protein binding[J]. Proc Natl Acad Sci U S A, 2023, 120(20):1-9.
[17]Curth U, Genschel J, Urbanke C, et al. In vitro and in vivo function of the C-terminus of Escherichia coli singlestranded DNA binding protein[J]. Nucleic Acids Res,1996, 24(14):2706-2711. DOI:10. 1093/nar/24. 14. 2706.
[18]Harrison ML, Desaulniers MA, Noyce RS, et al. The acidic C-terminus of vaccinia virus I3 single-strand binding protein promotes proper assembly of DNA–protein complexes[J]. Virology, 2016, 489:212-222.DOI:10. 1016/j. virol. 2015. 12. 020.
基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250391
中图分类号:Q936
引用信息:
[1]郭宴菲,孙宾莲,付艳.一种巨病毒单链DNA结合蛋白Crov372的功能鉴定[J].病毒学报,2026,42(03):801-811.DOI:10.13242/j.cnki.bingduxuebao.250391.
基金信息:
高致病性病毒与生物安全全国重点实验室自主部署项目(项目号：E5GF100103)，题目：高致病性病毒新型免疫及干预策略~~
2026-05-07
2026-05-07
2026-05-07