【作者】 潮礼君；

【导师】 陈应华；

【作者基本信息】 清华大学 ， 生物学， 2013， 博士

【摘要】 人类免疫缺陷病毒（HIV-1）跨膜蛋白gp41在病毒侵染靶细胞的膜融合过程中发挥了关键作用，是膜融合抑制剂研究中的重要靶点。此外，gp41还能够通过与靶细胞内某些蛋白发生相互作用，调控细胞的某些生理功能。在前期的研究中，我们通过酵母双杂交技术从人骨髓cDNA文库中筛选到一个HIV-1gp41的潜在结合蛋白POB1。基于POB1具有抑制HIV-1病毒介导的膜融合活性，本论文将POB1作为潜在的病毒抑制剂进行了相关研究，实验证明POB1具有抑制病毒感染的能力。在酵母双杂交实验的基础上，我们采用多种生化技术手段验证了HIV-1gp41和POB1的结合能力。结果表明，POB1通过C端螺旋区（C60）与gp41相互作用，而gp41的六螺旋结构及其包含的N末端七重复序列（NHR）与POB1均有较强的结合能力。我们将C60作为潜在的膜融合抑制剂进行了一系列研究。C60不能抑制gp41融合活性六螺旋结构的形成，但是在合胞体实验和病毒感染实验中均显示出一定的抑制活性，对X4嗜性株HIV-1IIIB和R5嗜性株HIV-1Bal的半抑制浓度在微摩尔级。这些结果表明，C60的抑制机理可能与已知膜融合抑制剂的抑制机理不尽相同，这两者的异同有待进一步阐明。对C60抑制机理的深入研究，有助于我们进一步揭示病毒介导的膜融合过程的细节。我们对gp41与POB1相互作用的生理意义进行了进一步的研究。POB1蛋白在细胞内参与了EGF介导的内吞信号通路，目前已经报导存在POB1(1-521)和POB1(1-659)两种同源蛋白。我们克隆出了一个新的同源蛋白POB1(1-460)。实验结果表明gp41与POB1(1-460)在真核细胞内依然能够发生相互作用。HIV-1gp41的存在能够显著增强A431细胞和CHO细胞的内吞作用，而且增强的内吞作用能够促进不表达CD4受体的A431细胞对HIV-1假病毒颗粒的摄入，这可能导致HIV-1病毒感染的增强。一个可能的推测是gp41所起的作用与EGF介导的信号类似，能够作为一个骨架蛋白招募参与内吞过程的POB1、epsin等蛋白，持续激活内吞通路。以上推测还需要后续实验证实。更多还原

【Abstract】 The HIV-1envelope glycoprotein gp41plays a crucial role in the retroviral fusionprocess, and it is a very important target for membrane fusion inhibitor. Gp41can alsoregulate some cellular functions by interacting with host proteins. In the previous study,we screened human bone marrow cDNA library by a yeast-two-hybrid system, andidentified POB1as a potential binding protein to gp41. Considering the finding thatPOB1could inhibit the HIV-1mediated membrane fusion, we investigated the functionof POB1as being a potential membrane fusion inhibitor. The results showed that POB1exhibited inhibitory activity against HIV-1infection.We used a variety of biochemical techniques to verify the binding capacitybetween HIV-1gp41and POB1. The experimental results showed that the C-terminalhelical region (C60) of POB1bound strongly to the gp41six-helix bundle (6-HB), or tobe more precise, the N-terminal heptad repeat (NHR) of gp41. C60exhibited inhibitoryactivity against the HIV-1Env-mediated syncytium formation and virus infection, andthe half maximal inhibitory concentration (IC50) values were in the micromolar range.Unlike traditional membrane fusion inhibitors, C60did not block the gp416-HBformation, which implied that the inhibition mechanism of C60might not be the sameas other fusion inhibitors. The differences between these two inhibition mechanismsneed to be further explained and the further study on the mechanisms will be helpful toimprove our understanding of the viral membrane fusion process.It was reported that POB1was involved in the EGF/insulin-induced endocyticpathway. We cloned a new POB1homology, POB1(1-460), and HIV-1gp41could stillbe able to interact with POB1(1-460) in293T cells. We also observed significantenhancement of EGF internalization in A431cells and CHO cells expressing gp41, andthe uptake of HIV-1virions in the CD4-negative A431cells was also increased, whichmight help more HIV-1virions to enter into target cells to establish effective infection.HIV-1gp41could play a role in EGF signaling and recruit proteins in the endocyticpathway, such as POB1, epsin, to activate the process of endocytosis. The hypothesisneeds further experiments to confirm.更多还原