基于代谢工程与生物正交反应的病毒原位标记技术研究
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摘要
示踪病毒在细胞内的感染路径对探究病毒的感染机制具有重要意义,病毒标记是实现病毒示踪的关键。针对目前病毒标记方法中存在的不足,该文提出了一种基于代谢工程的病毒原位生物正交标记技术。脂质/氨基酸/单糖的叠氮衍生物经细胞代谢将叠氮基团修饰在病毒表面,被修饰病毒粒子识别结合宿主细胞后与携带二苯基环辛炔(DBCO)的荧光探针经生物正交反应连接从而实现病毒标记。结果显示,叠氮基团在囊膜病毒和非囊膜病毒表面都有很好的修饰效果,并且在原位生物正交中携带二苯基环辛炔基团的荧光探针可快速捕捉到叠氮修饰的病毒粒子实现病毒标记。该标记方法不仅操作简单,而且适用于囊膜病毒与非囊膜病毒的标记。
Tracking virus infection pathway in cells is of great significance for understanding the viral infection mechanism, and the successful labeling on viruses is the key to achieve viruses tracking. In this study, we proposed the in situ bioorthogonal strategy based on the bioorthogonal chemistry and metabolic engineering for overcoming defects of traditional virus labeling methods. Azido motifs were incorporated into virions through cell metabolism with lipid/amino acid/monosaccharide azide derivatives, and the modified virions were in situ labeled with dibenzocyclooctyne-functionalized fluorescent probes through the bioorthogonal reaction. The results show that viruses incorporated with azide motifs could be quickly and effectively captured by fluorescent probes to achieve viruses in situ labeling. Significantly, the in situ bioorthogonal labeling strategy is a general method for enveloped and non-enveloped viruses labeling with simple operations.
Tracking virus infection pathway in cells is of great significance for understanding the viral infection mechanism, and the successful labeling on viruses is the key to achieve viruses tracking. In this study, we proposed the in situ bioorthogonal strategy based on the bioorthogonal chemistry and metabolic engineering for overcoming defects of traditional virus labeling methods. Azido motifs were incorporated into virions through cell metabolism with lipid/amino acid/monosaccharide azide derivatives, and the modified virions were in situ labeled with dibenzocyclooctyne-functionalized fluorescent probes through the bioorthogonal reaction. The results show that viruses incorporated with azide motifs could be quickly and effectively captured by fluorescent probes to achieve viruses in situ labeling. Significantly, the in situ bioorthogonal labeling strategy is a general method for enveloped and non-enveloped viruses labeling with simple operations.
引文
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[26]Pan H,Yao X,Chen W,et al.Dissecting complicated viral spreading of enterovirus 71using in situ biorthogonal fluorescent labeling[J]Biomaterials,2018,181(7):199-209.
[27]Zhao X,Shen Y,Adogla EA,et al.Surface labeling of enveloped virus with polymeric imidazole ligand-capped quantum dots via the metabolic incorporation of phospholipids into host cells[J]Journal of Materials Chemistry B,2016,4(14):2421-2427.
[28]Pan H,Zhang P,Gao D,et al.Noninvasive visualization of respiratory viral infection using biorthogonal conjugated near-infrared-emitting quantum dots[J].ACS Nano,2014,8(6):5468-5477.
[2]Wang IH,Burckhardt CJ,Yakimovich A,et al Imaging,tracking and computational analyses of virus entry and egress with the cytoskeleton[J]Viruses,2018,10(4):166-180.
[3]王亚林,仇华吉,孙元.疱疹病毒的示踪技术:看到了什么?还能看到什么?[J].生物工程学报2018,34(11):1721-1733.
[4]梁振普,王彩平,张小霞,等.单颗粒示踪技术及其在病毒侵染机制研究中的应用[J].病毒学报2017,33(4):638-645.
[5]Huang LL,Xie HY.Progress on the labeling and single-particle tracking technologies of viruses[J]The Analyst,2014,139(13):3336-3346.
[6]McDonald D,Vodicka MA,Lucero G,et al Visualization of the intracellular behavior of HIV in living cells[J].The Journal of Cell Biology,2002159(3):441-452.
[7]Greber UF,Way M.A superhighway to virus infection[J].Cell,2006,124(4):741-754.
[8]Chen J,Rahman SA,Nikolaitchik OA,et al.HIV-1RNA genome dimerizes on the plasma membrane in the presence of gag protein[J].Proceedings of the National Academy of Sciences of the United States of America,2016,113(2):201-208.
[9]Tyagi S.Imaging intracellular RNA distribution and dynamics in living cells[J].Nature Methods,20096(5):331-338.
[10]Lakadamyali M,Rust MJ,Babcock HP,et al Visualizing infection of individual influenza viruses[J].Proceedings of the National Academy of Sciences of the United States of America,2003100(16):9280-9285.
[11]Lakadamyali M,Rust MJ,Zhuang X.Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes[J].Cell,2006,124(5):997-1009.
[12]Rust MJ,Lakadamyali M,Zhang F,et al.Assembly of endocytic machinery around individual influenza viruses during viral entry[J].Nature Structural&Molecular Biology,2004,11(6):567-573.
[13]Joo KI,Lei Y,Lee CL,et al.Site-specific labeling of enveloped viruses with quantum dots for single virus tracking[J].ACS Nano,2008,2(8):1553-1562.
[14]Huang BH,Lin Y,Zhang ZL,et al.Surface labeling of enveloped viruses assisted by host cells[J].ACSChemical Biology,2012,7(4):683-688.
[15]Li J,Chen PR.Development and application of bond cleavage reactions in biorthogonal chemistry[J].Nature Chemical Biology,2016,12(3):129-137.
[16]Krall N,Da Cruz FP,Boutureira O,et al.Siteselective protein-modification chemistry for basic biology and drug development[J].Nature Chemistry,2016,8(2):103-113.
[17]Johnson JA,Lu YY,Van Deventer JA,et al Residue-specific incorporation of non-canonical amino acids into proteins:recent developments and applications[J].Current Opinion in Chemical Biology,2010,14(6):774-780.
[18]Salic A,Mitchison TJ.A chemical method for fast and sensitive detection of DNA synthesis in vivo[J]Proceedings of the National Academy of Sciences of the United States of America,2008,105(7):2415-2420.
[19]Patterson DM,Nazarova LA,Prescher JA.Finding the right(biorthogonal)chemistry[J].ACSChemical Biology,2014,9(3):592-605.
[20]Laughlin ST,Bertozzi CR.Metabolic labeling of glycans with azido sugars and subsequent glycanprofiling and visualization via staudinger ligation[J].Nature Protocols,2007,2(11):2930-2944.
[21]Huang LL,Lu GH,Hao J,et al.Enveloped virus labeling via both intrinsic biosynthesis and metabolic incorporation of phospholipids in host cells[J].Analytical Chemistry,2013,85(10):5263-5270.
[22]Pan H,Li WJ,Yao XJ,et al.In situ bioorthogonal metabolic labeling for fluorescence imaging of virus infection in vivo[J].Small,2017,13(17):196-214.
[23]Pan H,Zhang Y,Luo Z,et al.Autophagy mediates avian influenza H5N1 pseudotyped particleinduced lung inflammol/lation through NF-kappaBand p38 MAPK signaling pathways[J].American Journal of Physiology.Lung Cellular and Molecular Physiology,2014,306(2):183-195.
[24]Zheng LL,Yang XX,Liu Y,et al.In situ labelling chemistry of respiratory syncytial viruses by employing the biotinylated host-cell membrane protein for tracking the early stage of virus entry[J].Chemical Communications(Cambridge,England)2014,50(99):15776-15779.
[25]Hao J,Huang LL,Zhang R,et al.A mild and reliable method to label enveloped virus with quantum dots by copper-free click chemistry[J]Analytical Chemistry,2012,84(19):8364-8370.
[26]Pan H,Yao X,Chen W,et al.Dissecting complicated viral spreading of enterovirus 71using in situ biorthogonal fluorescent labeling[J]Biomaterials,2018,181(7):199-209.
[27]Zhao X,Shen Y,Adogla EA,et al.Surface labeling of enveloped virus with polymeric imidazole ligand-capped quantum dots via the metabolic incorporation of phospholipids into host cells[J]Journal of Materials Chemistry B,2016,4(14):2421-2427.
[28]Pan H,Zhang P,Gao D,et al.Noninvasive visualization of respiratory viral infection using biorthogonal conjugated near-infrared-emitting quantum dots[J].ACS Nano,2014,8(6):5468-5477.