【作者】 张哲；

【导师】 魏伟；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2021， 博士

【摘要】 研究背景及目的:人类肠道病毒（HEVs）为小核糖核酸病毒科肠道病毒属,包括脊髓灰质炎病毒、柯萨奇病毒、埃可病毒和新型肠道病毒等,都属于肠道病毒A-D组成员。多种肠道病毒在全球暴发流行,已成为严重的公共卫生问题,如EV-A71和EV-D68。类泛素化Neddylation是一类重要的蛋白翻译后修饰,在胚胎发育与细胞分裂增殖等生理过程中具有关键调控作用。与泛素化修饰途径相似,NEDD8蛋白在E1激活酶（NAE1）、E2连接酶（UBE2M和UBE2F）和E3连接酶的多步级联酶促反应下,共价结合到底物蛋白质,参与调节蛋白质的稳定性与功能。越来越多的证据表明,不同病毒经常靶向Neddylation修饰通路调节宿主内环境以促进病毒复制。因此,我们推测Neddylation修饰通路可能对肠道病毒复制至关重要,并可能存在抗病毒治疗靶点。NAE1小分子机制剂MLN4924已经作为抗肿瘤药物在多种癌症的I/II期临床试验中表现出显著的疗效。此外,MLN4924亦是一种有效的抗病毒候选药物,对多种人类病毒具有显著的抗病毒活性。因此,我们旨在阐明Neddylation修饰通路对肠道病毒复制的影响,并探索MLN4924作为抗肠道病毒感染的候选抗病毒药物的潜在临床应用价值。研究方法:1、MLN4924对EV-A71复制的影响:（1）EV-A71感染（MOI 0.01）不同浓度的MLN4924（剂量分别为0.02μM、0.05μM、0.1μM、0.5μM、1μM和2μM）或DMSO处理的RD细胞,感染48 h后观察细胞病变效应、计算上清病毒滴度TCID50、免疫印迹法检测病毒VP1蛋白表达。（2）MLN4924细胞毒性检测和EC₅₀:将细胞在含有不同浓度的MLN4924（0-20μM）培养基中培养48 h,使用MTS法评估细胞增殖和细胞毒性。EV-A71感染不同浓度MLN4924处理的RD细胞后48 h,检测病毒RNA的水平,计算EC₅₀。（3）MLN4924对病毒吸附和进入阶段的影响:将RD细胞分别用MLN4924或DMSO处理,然后与EV-A71在4℃或37℃下孵育2 h,以促进病毒附着或进入,检测病毒RNA水平。（4）采用qRT-PCR检测MLN4924处理组和DMSO对照组特定时间点细胞内和上清的病毒RNA浓度,免疫印迹法检测EV-A71 VP1蛋白表达。（5）为了明确MLN4924对EV-A71的抑制作用在不同类型细胞中的广谱性,我们又另外在人结肠癌上皮细胞HT-29中和肺癌细胞系A549中进行了验证。EV-A71感染MLN4924或DMSO处理的HT-29和A549细胞,感染后48 h观察细胞病变效应、检测病毒RNA水平、VP1蛋白表达及上清病毒滴度。2、MLN4924抑制病毒复制的作用机制研究:通过sh RNA慢病毒包装技术构建稳定敲低NAE、E2结合酶UBE2M和UBE2F的RD细胞系,感染相同MOI的EV-A71病毒,48 h后观察细胞病变效应、台盼蓝染色细胞计数计算细胞存活率、qRT-PCR检测病毒RNA水平、免疫印迹法检测病毒VP1蛋白表达以及TCID50计算上清病毒滴度。3、MLN4924对EV-D68复制的影响:用MOI为0.1的EV-D68感染MLN4924或DMSO处理的HEK293T细胞或者A549细胞,观察细胞病变效应、RNA复制水平、VP1表达及上清病毒滴度。结果:1、MLN4924抑制肠道病毒EV-A71复制:（1）MLN4924抑制RD细胞中EV-A71细胞病变效应,并显著降低上清子代病毒产生。此外,EV-A71 VP1蛋白表达在MLN4924处理的细胞中呈剂量依赖性降低。MLN4924抑制RD细胞EV-A71 RNA复制的EC₅₀值约为0.011μM。MTS检测结果表明,MLN4924即使浓度高达20μM,也没有观察到明显的细胞毒性或细胞增殖下降。（2）在吸附和进入实验中,我们比较了药物处理组和DMSO组RNA复制水平,发现两组均没有明显差异。说明MLN4924对EV-A71病毒吸附和入侵没有显著影响。随后,通过对感染后特定时间点细胞内和上清的EV-A71 RNA复制进行分析,与DMSO对照组细胞相比,MLN4924处理的RD细胞裂解液和上清液中的病毒RNA积累明显减少。接下来,我们检测了MLN4924或DMSO作用下,EV-A71感染RD细胞后VP1蛋白的表达情况。在MLN4924和DMSO处理的细胞中,VP1蛋白在8 hpi时均可检测到表达。在随后的时间点,MLN4924组细胞和上清中观察到VP1蛋白的表达显著减少。（3）MLN4924抑制HT-29细胞中EV-A71 RNA复制、VP1蛋白表达以及子代病毒产生。在A549细胞中,我们也观察到MLN4924显著抑制EV-A71细胞病变效应、VP1蛋白表达以及子代病毒颗粒产生。2、EV-A71的复制在稳定表达的RD细胞系sh NAE、sh UBE2M或sh UBE2F中受到抑制:下调NAE、UBE2M和UBE2F显著降低了病毒细胞病变效应、RNA复制、VP1蛋白表达以及感染性病毒的产生。3、MLN4924抑制肠道病毒EV-D68复制:为了验证MLN4924对其他肠道病毒如EV-D68是否也具有抗病毒活性,我们在HEK293T和A549中也进行了相关实验。MLN4924可以使被EV-D68感染的HEK293T和A549细胞病变减轻,还能显著降低病毒RNA复制、VP1蛋白表达水平以及感染性病毒的产生。结论:1、不同细胞系中低浓度范围的MLN4924具有抑制EV-A71病毒复制的能力,说明MLN4924是一种安全有效的、抗肠道病毒复制的抑制剂。2、MLN4924对EV-A71病毒的吸附和进入过程没有影响,对病毒的抑制作用发生在进入细胞之后的复制阶段。3、EV-A71的复制在稳定表达的RD细胞系sh NAE、sh UBE2M或sh UBE2F中受到抑制,说明Neddylation修饰对肠道病毒的生命周期至关重要,宿主蛋白NAE、UBE2M和UBE2F是肠道病毒感染的关键因素。4、MLN4924能有效抑制EV-D68复制,证明了MLN4924具有作为抗肠道病毒感染的候选抗病毒药物的潜力。更多还原

【Abstract】 Background and objective: Human enteroviruses（HEVs）,a genus Enterovirus in the family Picornaviridae,comprise polioviruses,coxsackieviruses,echoviruses,and the enterovirus subgroups,which are all belong to four species,Enterovirus A–D.Several enteroviruses,such as EV-A71 and EV-D68,have emerged as serious public health problems in global outbreaks.Neddylation is an important post-translational modification of proteins and plays a key regulatory role in physiological processes such as embryonic development and cell division and proliferation.Similar to the ubiquitination pathway,NEDD8 protein covalently binds to the target protein in a multi-step cascade of E1 activation enzyme（NAE1）E2 ligase（UBE2M and UBE2F）and E3 ligase,and participates in the regulation of protein stability and function.Accumulating evidence suggests that different viruses frequently target Neddylation signaling pathways and modulate the intracellular environment of the host to enhance virus replication inrecently years.Therefore,we speculate that the Neddylation of the modified pathway may be crucial to the replication of EV-A71 and may be a target for antiviral treatment.NAE1 inhibitor MLN4924 has shown significant efficacy as an antineoplastic agent in phase I/II clinical trials in a variety of cancers.Besides,MLN4924 has been found to be an optimal antiviral drug candidate against diverse human viruses.Herein,we set out to investigate the effects of the Neddylation pathway on enterovirus replication and to explore the potential clinical value of MLN4924 as an antiviral drug candidate against enterovirus infection.Methods: 1.The effect of MLN4924 on EV-A71 replication:（1）Human rhabdomyosarcoma RD cells were treated with MLN4924 at various concentrations（0.02,0.05,0.1,0.5,1 and 2 μM）and challenged with EV-A71（multiplicity of infection,MOI of 0.01）.Cytopathic effect（CPE）was observed at 48 h post-infection（hpi）,supernatants were collected and viral titers were calculated as TCID50.The protein expression levels of enterovirus VP1 protein were detected using immunoblotting.（2）The cytotoxicity of MLN4924 was determined in mock-infected RD cells.Cells were cultured in media containing a range of MLN4924 concentrations（0–20 μM）,and cell proliferation and cytotoxicity were evaluated using the MTS assay.RD cells were treated with different concentrations of MLN4924 and collected 48 h after EV-A71 infection.Viral RNA levels was detected using q RT-PCR,the curve was drawn,and EC₅₀ was calculated.（3）RD cells were treated with either MLN4924 or DMSO and then incubated with EV-A71 for 2 h at 4 °C or 37 °C to facilitate virus attachment or entry,respectively.q RT-PCR was used to detect viral RNA（v RNA）.（4）The concentrations of intracellular and supernatant viral RNA at specific time points post-infection were determined using q RT-PCR,the effect of MLN4924 treatment on EV-A71 VP1 protein expression at specific time points post-infection was detected using immunoblotting.（5）We performed the same experiments in Human colorectal carcinoma cells HT-29 and human lung cancer cells A549.HT-29 and A549 were treated with MLN4924 and challenged with EV-A71.Cytopathic effect（CPE）was observed at 48 h post-infection（hpi）,supernatants were collected and viral titers were calculated as TCID50.The protein expression levels of enterovirus VP1 protein were detected using immunoblotting.2.Study on the mechanism of MLN4924 inhibiting EV-A71 replication: We generated stable knockdown RD cell lines using sh RNA targeting endogenous E1（NAE1）and E2（UBE2M or UBE2F）enzymes.The cells were then challenged with EV-A71.CPE was observed in RD cells at 48 hpi.The survival rates of infected cells were determined using trypan blue staining.Viral RNA was determined using q RT-PCR at 48 h post-infection.The expression of VP1 was detected using immunoblotting.Supernatants were collected at 48 hpi,and viral titers were calculated as TCID50.3.The effect of MLN4924 on EV-D68 replication: MLN4924-or DMSO-treated HEK293 T cells and A549 cells were infected with EV-D68.CPE was observed 48 h post-infection,viral RNA（v RNA）was detected using q RT-PCR,EV-D68 VP1 expression were detected using immunoblotting,and viral titer of supernatant was calculated using TCID50 assay.Results: 1.MLN4924 suppresses EV-A71 replication:（1）MLN4924 treatment markedly inhibited EV-A71 CPE and significantly reduced the titers of progeny virus in the supernatant.In addition,EV-A71 VP1 protein expression decreased in a dose-dependent manner in MLN4924-treated cells.MLN4924 inhibited EV-A71 RNA replication in RD cells at an EC₅₀ value of approximately 0.011 μM.No significant cytotoxicity or decrease in cell proliferation was observed in MLN4924-treated cells,even at concentrations as high as 20 μM,as determined using the MTS assay.（2）We found that MLN4924 treatment did not significantly affect EV-A71 virus attachment and entry.Time-dose analysis of EV-A71 RNA replication indicated a strong decrease in the accumulation of viral RNA in the cell lysate and in the supernatant of MLN4924-treated RD cells,when compared to that in DMSO-treated cells.We next analyzed VP1 protein expression in RD cells infected with EV-A71 either in the presence of 0.5 μM MLN4924 or DMSO over a time course.VP1 protein expression was detectable at 8 hpi in both MLN4924-and DMSO-treated cells.At later time points,a dramatic reduction in VP1 protein expression was observed in both the cell-associated and supernatant-associated fractions of MLN4924-treated cells as compared to the DMSO-treated controls.（3）MLN4924 inhibited EV-A71 replication in HT-29 and A549: MLN4924 significantly reduced EV-A71 CPE,RNA replication,VP1 protein expression and progeny virus production.2.EV-A71 replication was inhibited in the stable knockdown RD cell lines sh NAE,sh UBE2 M or sh UBE2M: These results showed that the downregulation of NAE1,UBE2 M,and UBE2 F dramatically reduced enterovirus CPE,the replication of viral RNA,the expression of VP1 protein and the production of infectious virions,as compared with those in control cells.3.MLN4924 inhibits EV-D68 replication: MLN4924 treatment significantly blocked EV-D68 CPE,VP1 protein expression and infectious virion production in HEK293 T.MLN4924 treatment potently inhibited EV-D68 replication in human lung carcinoma A549 cells.Conclusions: 1.MLN424 can inhibit EV-A71 virus replication at low concentrations in different cell lines,suggesting that MLN4924 is a safe and effective inhibitor against enterovirus replication.2.MLN4924 treatment did not significantly affect EV-A71 virus attachment and entry,indicating that MLN4924 suppresses EV-A71 replication at a post-entry step.3.EV-A71 replication was inhibited in the stable knockdown RD cell lines sh NAE,sh UBE2 M or sh UBE2 M,suggesting that Neddylation pathway is a determinant factor for supporting EV-A71 replication and NAE,UBE2 M and UBE2F are required for enterovirus infection.4.MLN4924 treatment potently inhibited EV-D68 replication,which proves that MLN4924 has the potential as a broad-spectrum antiviral drug candidate against enterovirus infection.更多还原