【作者】 王晓丹；

【导师】 于湘晖； 孔维； 杨茂君；

【作者基本信息】 吉林大学 ， 微生物学， 2013， 博士

【摘要】 Vif（viral infectivity factor）基因编码的蛋白作为HIV-1（AIDS病原体）的六个辅助因子之一，是一个高碱性的分子量为23kDa的磷蛋白，它在逆转录病毒生命周期的早期步骤和晚期步骤中发挥功能。HIV-1对Vif的需求受产病毒细胞的类型限制，即Vif对于HIV-1在非允许型细胞系中产生有传染效力的子代病毒和有效感染其他细胞至关重要，缺失了Vif的HIV-1能在允许细胞中而不能在非允许细胞有效复制。而特异性表达于非允许细胞中的APOBEC3G（CEM15）提供了这个非允许特性。它在2002年被鉴定为抗HIV-1的宿主因子。它主要作用于病毒生命周期的早期，能够包装进入多种逆转录病毒（HIV-1/HIV-2，马传染性贫血病毒，多种猴免疫缺陷病毒等等），随即启动大规模的针对逆病毒新生负义链cDNA的脱氨基作用，借此引发病毒基因组的高突变，导致病毒蛋白发生错义突变或提前终止病毒蛋白转录。Vif能够对A3G的抗病毒作用进行中和，它通过将APOBEC3G募集到人体内的CBF-β-ElonginB-ElonginC-Cullin5-Rbx（CBF-b-EloB-EloC-Cul5-Rbx）复合物上形成E3泛素连接酶上介导其降解。Vif用于募集E3复合物的功能位点已经被研究得比较透彻，Vif的N端包含了与CBF-β和A3G相互作用的位点，Vif的C端包含一个用于结合EloB-EloC的SOCS-box基序，一个用于结合Cul5的HCCH锌指基序。E3复合物中的每个组分对于降解A3G都不可或缺。Vif在复合物中作为A3G底物的接头分子，新发现的CBF-β通过调节Vif折叠作为调节子，EloB-EloC也是调节亚基而Cul5为基架蛋白。尽管关于Vif各区域功能的报道与日俱增，Vif结构仍未能得到彻底解析，2008年Vif SOCS基序的结构得到解析然而更多的Vif结构信息仍有待探索。作为新发现的调节亚基，CBF-β乃至整个E3的功能机制仍有待完善。本论文针对Vif-EloB-EloC复合物结构以及该复合物各组分间的相互作用机制展开了研究。我们对该复合物的组成蛋白进行多种截短和组合，通过X-蛋白晶体衍射技术试图进一步解析Vif结构及其与EloB-EloC的相互作用界面。以期获得最直接的作用机制信息指导治疗药物设计。通过蛋白共折叠可溶性分析，RNA沉默，免疫共沉淀，体内稳定性，关键基序突变等多种分子生物学手段分析E3复合物各组分相互作用机制。在蛋白复合物结构研究部分，应用改良载体PMR1以及具有多个读码框，能够同时表达多种蛋白的原核表达载体pST39作为复合物表达载体，我们构建，表达并纯化了HIV-1Vif蛋白单体及其六种复合物：PMR1-Vif；pST-EloC-EloB-vif；pST-EloC-EloB-Vif-Cul511-191； pST-EloC-EloB-Vif-Cul51-159；pST-EC-EB-Vif-Cul51-230； pST-EloCΔN17-EloB-VifΔN97； pST-EloCΔN17-EloBΔC20-VifΔN97。均获得可供晶体生长的可溶蛋白，其中Vif单体，EloCΔN17-EloB-VifΔN97，EloCΔN17-EloBΔC20-VifΔN97获得了纯度在95%以上的寡聚蛋白，Vif单体蛋白获得衍射为1.6的晶体，然而由于缺乏结构模型，硒代衍生物结晶失败而无法解析。随后我们对纯化得到的EloCΔN17-EloB-VifΔN97蛋白复合物进行了温和的胰蛋白酶剪切处理，去掉折叠松散，肽链骨架灵活度过高的区段，使得该复合物结晶概率提高。得到分辨率2.4可供结构解析的晶体，然而由于该晶体为孪晶难以成功解析，现正对已获得的结晶条件进行结晶温度优化，沉淀剂浓度优化，PH值优化等一系列优化，以期提高晶体质量，得到成功解析。对酶切处理前后的复合物VifΔN97-EB-ECΔN17进行免疫印迹法分析，发现酶切后EloB，EloC分子量没有明显改变，而Vif的分子量显著减小到4-5kDa，通过液相串联质谱联用分析认为酶切后Vif片段约为残基123-168，说明该区段折叠紧实因而防止了胰蛋白酶的剪切，揭示了Vif与EloB-EloC共折叠时C端的折叠核心。而对酶切前后复合物进行浊度分析等物理化学性质分析发现酶切后的复合物性质与酶切前有所不同，酶切后的蛋白热稳定性更优于酶切前。通过该部分研究，我们对从一个新的角度分析了Vif C端的折叠。在复合物功能分析部分，为了深入研究Vif-EloB-EloC-CBF-β复合物成员的功能与组装，我们沉默了内源EloB的表达，发现以下结论：（1）和以前一些研究结果相同，沉默了内源的EloB后，EloC的内源表达量将随之下降，再次肯定了EloB和EloC是作为专性复合物存在的。（2）EloB表达下调后，影响了Vif对A3G的降解，使得Vif阻止A3G包装进病毒的功能下降，从而使Vif拮抗A3G抗病毒的能力下降。（3）研究这一现象的机理，我们发现EloB表达下调后，会显著削弱CBF-β, Cul5与Vif的结合而不影响A3G与Vif的结合。（4）我们使用了VifΔSLQ这一能够削弱EloB-EloC结合的Vif突变体，发现其结合CBF-β的能力显著弱于野生型Vif。（5）我们构建了删除Vif结合位点的EloBΔC34突变体，发现该突变体的过表达能够削弱Vif对A3G的降解能力且该削弱为剂量依赖性的，也能够削弱Vif对A3G抗病毒功能的拮抗。（6）使用删除了EB作用位点的VifΔPPL突变体，发现该突变能够削弱CBF-β与Vif的作用。（7）在大肠杆菌内通过共表达可溶性验证发现，只有EloB-EloC存在的情况下，才能够通过辅助Vif折叠使得Vif与CBF-β大量存在于上清中。我们由以上发现得到结论：Vif与EloB-EloC的相互作用对于Vif与CBF-β的相互作用十分重要。此外我们还发现EloB及其泛素样结构域的过表达能够显著稳定Vif在细胞内的蛋白水平，该发现丰富了泛素样分子的功能多样性。本研究为解析Vif-EloB-EloC复合物相互作用的结构界面提供了基础，延伸了我们对Vif-Cul5E3复合物功能机制的理解，为HIV的药物治疗提供了新的可能性。更多还原

【Abstract】 HIV-1is the causative agent of the acquired immunodeficiency syndrome (AIDS).As one of the six HIV-1accessory gene, Vif encodes a highly basic23kDaphosphoprotein that functions in both early and late steps of the retroviral life cycle. Vif(virion infectivity factor) is necessary for the production of infectious virus and efficientviral infection in non-permissive cell lines. The requirement for the vif gene in efficientHIV-1replication is tightly linked to the phenotype of the virus-producing cell. HIV-1lacking vif can efficiently replicate in permissive cell lines but not in non-permissivecells. CEM15, now usually known as human APOBEC3G(A3G) which is present onlyin nonpermissive cells confering a nonpermissive phenotype, was identified as ananti-HIV host factor in2002. It can inhibit early stages of the retrovirus life cycle.APOBEC3G functions by incorporation into various budding viruses (includingHIV-1/HIV-2, equine infectious anemia virus and simian immunodeficiency viruses etal) and subsequently triggers hypermutations by massive deamination within thenascent retroviral minus-strand cDNA, causing many missense and nonsense codonchanges.Vif antagonizes the antiviral activity of human cytidine deaminase APOBEC3proteins confering the non-permissive phenotype by tethering A3G to theVif-CBF-β-ElonginB-ElonginC-Cullin5-Rbx (Vif-CBF-β-EloB-EloC-Cul5-Rbx) E3complex to induce its proteasomal degradation. The functional domains that Vif utilizesto recruit the E3complex have been well studied: The N-terminal region of Vif containsthe main sites involved in binding of CBF-β and APOBEC3proteins; while itsC-terminal domain contains a so-called SOCS-box motif, which is responsible forbinding to the EloB-EloC complex, and a conserved HCCH zinc coordination site thatmediates selective binding to Cul5. Every component of the Vif-Cul5E3ligase is indispensable for degradation of APOBEC3proteins. Vif acts as a substrate adaptormolecule to bridge A3G with the Cul5E3ligase, and the newly found factor CBF-β isreported to be a unique regulator of Vif-Cul5E3ligase by promoting folding of Vif,EloB and EloC are also known as regulatory subunits, whereas Cul5functions as ascaffold protein. Though the biochemical data is rapidly accumulated, the structural dataon HIV-1Vif protein is insufficient so far. The crystal structure of the HIV-1VifBC-box-ElonginB-ElonginC complex has been determined in2008. More structuralinformation to investigate the relationship between the structure and the function of Vifis still waiting to be explored. CBF-β is a newly identified key regulator of Vif functionand more information is needed to further clarify its regulation mechanism.Our study focused on the structure of Vif-EloB-EloC and the mechanism of theinteractions among the components of Vif E3complex. We tried various ofVif-EloB-EloC truncations to attempt to further analyse Vif-EloB-EloC structure byX-ray. For the purpose of obtaining the most direct informations to guide thetherapeutic drug design, we comprehensively investigated the interactions of thecomponents of the Vif-Cul5E3ligase complex by using solubility analysis， RNAinterference, co-immunoprecipitation assay, cycloheximide stability assay, viralinfectivity assay and the critical motif mutation et al.In the structure analysis, by using a modified vector PMR1and a polycistronicco-expression plasmid pST39, We constructed and expressed Vif protein as well as aseries of soluble protein complex of EloC-EloB-Vif；EloC-EloB-Vif-Cul511-191；EloC-EloB-Vif-Cul51-159；EloC-EloB-Vif-Cul51-230；EloCΔN17-EloB-VifΔN97；EloCΔN17-EloBΔC20-VifΔN97in BL21E. coli cells. All of these proteins had beenisolated for growing crystals. The purities of Vif，EloCΔN17-EloB-VifΔN97complexand EloCΔN17-EloBΔC20-VifΔN97complex were even>95%. We obtained a singlecrystal of Vif which diffrated to1.6resolution, however, the structrue could not beresolved because there was not any known protein structural motif in Vif and becausethe failure of getting the selenomethionine-incorporated protein. Then we carried out mild trypsin treatment to the EloCΔN17-EloB-VifΔN97complex to remove thefractions with low compactness and high flexibility in the backbone. After the cleavagereaction, the cleaved complexes were isolated from the reaction mixture by a two-steppurification: Ion exchange-Gel filtration, for the purpose of getting compactly foldedglobulin complexes. This cleaved complex could be crystallized and the crystals diffractto2.40resolution. However, so far we are unable to determine the structure of thetrypsin-treated HisVifΔN97-EloB-EloCΔN17complex in this research because thediffraction data could not be indexed since the crystals are twin, consequently lacking ofthe data parameters. Refinements of the crystallization condition is in progress bychanging the crystallization temperature, concentration of precipitant, buffer pH, proteinsolution concentration, drop size, and additives in order to get single crystals.SDS-PAGE and Western blot analysis showed that after incubation with trypsin,HisVifΔN97had a decreased molecular weight about4-5kDa whereas ElonginB andElonginCΔN17had almost no changes. The Vif fragment in the cleaved complex wasthen identified by LC-MS/MS, the result indicated the peptide of Vif C-terminal123-168or more amino acid residues which in complex with ElonginB and ElonginCwas well folded or bound compactly to ElonginB-ElonginC therefore shielded frommild tryptic attack, revealing the folding core in Vif C-terminal. We also found that thecleaved complex showed a increase in thermal stability by using turbidity analysis,analyzing the folding of Vif C-terminal domain from a new perspective.About function analysis of the E3complex, in an attempt to comprehensivelyinvestigate the components functions of the Vif-CBF-β-EloB-EloC-Cul5E3ligase, weknocked down endogenous EloB expression by using siRNA. Major findings were asfollows:(1) We found it was consistent with the existing results obtained by Hwang thatthe levels of EloC were greatly reduced in EloB-silencing cells, confirmed again thatEloB formed an obligate heterodimer with EloC.(2) Knock-down of endogenous EloBexpression impaired Vif-induced A3G degradation and the ability of Vif to block theincorporation of A3G into the budding virus. Silencing of endogenous EloB could also impair the ability of Vif to neutralize the antiviral activity of A3G.(3) To explore themechanism used by EloB to affect Vif function, we examined the interactions of HIV-1Vif with its substrate A3G and its cognate partners, endogenous Cul5, EloB/EloC andCBF-β in the condition of EloB silencing. We found that EloB silencing (together withEloC) could reduce Vif binding to CBF-β and Cul5, whereas binding to A3G seemednot be affected.(4) We used a VifΔSLQ mutant which dramatically impairsVif-EloB-EloC binding and found that there was almost no CBF-β interaction withVif-SLQ.(5) We constructed a mutant with a deletion of the34amino acids in theC-terminal tail which was reported to interact with Vif, EBΔC34. The Vif-mediateddepletion of A3G was impaired by co-expression of EBΔC34, and this effect ofEBΔC34on Vif function was dose-dependent, the presence of EBΔC34also interferedwith the ability of Vif to counteract the A3G antiviral activity.(6) Since EloB wasreported to interact with the PPLP motif of Vif, we wondered that whether thisinteraction could also affect CBF-β binding to Vif, to test this hypothesis, We used aVifΔPPL mutant and found that VifΔPPL indeed appeared to be less able to bindCBF-β.(7) We found that it was EloB-EloC but not CBF-β could greatly enhance thefolding of full-length Vif in Escherichia coli, and solubilities of Vif and CBF-β weresignificantly increased only when they were co-expressed with both EloB and EloC. Inconclusion, our results demonstrate that the interactions between HIV-1Vif and humanEloB-EloC are important for CBF-β binding to Vif. Moreover, we found that free EloBcan stabilize Vif via its UbH domain, further expanding the diversity of functions ofUbL proteins.Our studies provide a material basis for resolving the molecular structure of theVif-EloB-EloC interaction interface，extend the current understanding of the functionand mechanism of the Vif-CBF-β-Cul5E3ligase and offer new possibilities for therapeutic applications.更多还原