【作者】 刘勇；

【导师】 王炜；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2023， 硕士

【摘要】 Ⅰ型人类免疫缺陷病毒（Human Immunodeficiency Virus Type Ⅰ,HIV-1）是一种逆转录病毒。在其复制过程中,HIV-1以tRNA^Lys,3为引物,将其基因组RNA（Genomic RNA,gRNA）逆转录为DNA。因此,HIV-1需要将细胞中tRNA^Lys,3包装至病毒颗粒内,并使tRNA^Lys,3结合至gRNA 5’端非翻译区（5’Untranslated Region,5’UTR）的引物结合位点（Primer Binding Site,PBS）,进而启动逆转录。在病毒复制后期,tRNA^Lys,3与人类赖氨酸-tRNA合成酶（Lysyl-tRNA Synthetase,LysRS）形成复合物,并被一同包装至病毒颗粒中的。为了使tRNA^Lys,3结合至PBS,HIV-1 gRNA 5’UTR演化出了类tRNA结构（tRNA-like-structure,TLE）,可以与tRNA^Lys,3竞争性结合LysRS。这种竞争使tRNA^Lys,3从与LysRS形成的复合物中释放并结合至PBS区域。但5’UTR具有复杂的结构,并可以在不同构象和聚集形态间转换。目前还不清楚5’UTR的构象、聚集形态及各结构域对5’UTR与LysRS互作的影响。本文拟通过研究LysRS与HIV-1 5’UTR的相互作用,研究HIV-1的类tRNA结构参与逆转录的机制,为后续针对此过程的药物开发提供新的思路和方法。为了研究LysRS和5’UTR的相互作用,我们构建了体外实验体系。通过体外转录制备HIV-1 5’UTR及其不同形式的突变体RNA分子（以下称为5’L分子）,使这些RNA分子呈现出不同的构象和聚集状态。在原核表达系统中表达纯化了LysRS蛋白（实验中所用蛋白为LysRSΔN65,截去N端65个氨基酸以减弱LysRS与RNA的非特异性结合）。使用聚丙烯酰胺凝胶电泳（Polyacrylamide Gel Electrophoresis,PAGE）和尺寸排阻色谱（Size Exclusion Chromatography,SEC）验证它们化学性质的稳定性和构象的均一性。通过荧光偏振实验（Fluorescence Polarization,FP）探究不同5’L分子与LysRSΔN65的相互作用,明确相互作用过程中5’UTR上起关键作用的结构域。模拟细胞中5’UTR与tRNA^Lys,3竞争结合LysRS的过程,通过荧光偏振竞争实验验证5’L分子与tRNA^Lys,3竞争结合LysRSΔN65过程中5’UTR上起关键作用的茎环结构。研究发现,相比于二聚体,单体5’L分子与LysRSΔN65有着更强的亲和力,表明二聚化不利于两者的的结合。而相比于包装构象,翻译构象的5’L分子与LysRSΔN65的亲和力显著提高,提示构象变化在5’UTR与LysRS的相互作用过程中发挥关键作用。包装构象5’UTR的PBS和包装信号（Packaging Signal,Ψ）区域在结合过程中发挥关键作用,而TAR-Poly A茎环结构则不利于两者结合。包装构象5’UTR中含有Ψ区域的5’L分子与LysRS的结合较少受到盐浓度的影响。竞争实验表明含有PBS和Ψ区域的5’L分子能有效地与tRNA^Lys,3竞争结合LysRS。以上结果表明PBS和Ψ区域是5’UTR上与LysRS相互作用的关键区域。综上所述,5’UTR构象变化和5’UTR中PBS和Ψ区域在逆转录引物结合过程中发挥重要作用。这些结果为破坏逆转录引物与PBS结合进而干扰病毒复制奠定了理论基础,为后续针对此过程的药物设计提供了新的思路和方法。更多还原

【Abstract】 Human Immunodeficiency Virus Type Ⅰ（HIV-1）is a retrovirus.During its replication,HIV-1 uses tRNA^Lys,3 as a primer to reverse transcribe its genomic RNA（gRNA）into DNA.tRNA^Lys,3 therefore needs to be packaged into the viral particle in the cell,and to bind to the Primer Binding Site（PBS）at the 5’Untranslated Region（5’UTR）of the gRNA which initiates reverse transcription.During late viral replication,tRNA^Lys,3 forms a complex with human lysyl-tRNA synthetase（LysRS）and this complex is packaged into the viral particle.In order to make tRNA^Lys,3 bind to PBS,the HIV-1 gRNA 5’UTR has evolved a tRNA-like structure（TLE）that competes with tRNA^Lys,3 to bind LysRS.This competition allows tRNA^Lys,3 to be released from the complex formed with LysRS and to bind to the PBS region.However,the5’UTR has complex structure and can switch between different conformations and aggregation forms.It is not clear how the conformation,aggregation pattern and the structural domains of 5’UTR affect the interaction of 5’UTR with LysRS.In this paper,we intend to investigate the interaction between LysRS and HIV-1 5’UTR to explore the mechanism of the tRNA-like structure of HIV-1 involved in reverse transcription,and provide new ideas and methods for subsequent drug development targeting this process.To study the interaction between LysRS and 5’UTR,we constructed an in vitro experimental system.HIV-1 5’UTR and its different forms of mutant RNA molecules（hereafter referred to as 5’L molecules）were prepared by in vitro transcription,so that these RNA molecules can form different conformations and aggregation states.The LysRS protein was purified by expression in a prokaryotic expression system（the protein used in the experiments was LysRSΔN65,with the N-terminal 65 amino acids truncated to attenuate the non-specific binding of LysRS to RNA）.Their chemical stability and conformational homogeneity were verified using Polyacrylamide Gel Electrophoresis（PAGE）and Size Exclusion Chromatography（SEC）.The interaction of different 5’L molecules with LysRSΔN65 was investigated by fluorescence polarization（FP）to identify the structural domains on the 5’UTR that play a key role in the interaction process.Then We simulated the competition between 5’UTR and tRNA^Lys,3for LysRS binding in cells,and verified the structures of the stem loop on5’UTR that play a key role in the competition between 5’L molecules and tRNA^Lys,3 for LysRSΔN65 binding by fluorescence polarization competition experiments.It was found that the monomeric 5’L molecule has a stronger affinity for LysRSΔN65 than the dimer,suggesting that dimerization is detrimental to the binding to LysRSΔN65.The translational conformation of 5’L molecules showed significantly higher affinity for LysRSΔN65 compared to the packing conformation,suggesting that conformational changes play a key role in the interaction of 5’UTR with LysRS.The PBS and Packaging Signal（Ψ）regions of the 5’UTR play a key role to bind to LysRSΔN65,whereas the TAR-Poly A stem-loop structure is not conducive to the binding.The binding of 5’L molecules containing theΨregion in the monomeric packing conformation 5’UTR to LysRS was less affected by salt concentration.The above results suggest that the PBS andΨregions are the key regions for the interaction with LysRS on the 5’UTR.Taken together,the 5’UTR conformational changes and the PBS andΨregions in the 5’UTR play important roles in the reverse transcription primer binding process.These results provide a theoretical basis for disrupting the binding of reverse transcription primers to PBS and thus interfering with viral replication,and provide new ideas and methods for subsequent drug design targeting this process.更多还原