【作者】 樊博；

【导师】 蒋建东； 岑山； 彭宗根；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2010， 博士

【摘要】 病毒感染性疾病严重危害人类健康,以不同病毒酶蛋白为靶点的抑制剂联合治疗方案在临床抗病毒治疗中虽然能取得较好的治疗效果,但是长期用药后病毒易发生耐药性突变,对治疗效果造成严重影响。因此,寻找新的治疗策略是当下抗病毒药物研究工作的重点之一。随着病毒学研究的深入,发现病毒在复制过程中,会与多种宿主细胞蛋白发生相互作用,一方面,病毒利用特定的宿主细胞蛋白,以促进其复制,另一方面,人体的固有免疫机制通过部分特定功能蛋白在病毒生活周期的不同阶段以多种有效机制抑制病毒复制(这种蛋白称之为细胞限制因子)。因此,寻找与病毒复制相关,而宿主细胞生存非必需的蛋白有可能成为抗病毒药物的新靶点。第一部分：以往的研究显示,宿主细胞的分子伴侣热应激蛋白Heat stress cognate 70 (Hsc70,HspA8)广泛参与多种RNA和DNA病毒的复制。近期研究表明,Hsc70被包装进入丙型肝炎病毒(Hepatitis C Virus, HCV)病毒颗粒,可调节病毒感染性,并在子代病毒感染过程中帮助HCV RNA的释放。我们认为或许可以以Hsc70为靶点,探索抗HCV病毒新的治疗策略。我们首先建立在人肝Huh7.5细胞内用实时荧光定量RT-PCR技术检测胞内Hsc70 mRNA表达的药物筛选模型,通过筛选发现本研究所化学合成室合成的化合物IMB-DM122可以特异性地显著下调Hsc70 mRNA水平超过80%,因此我们将IMB-DM122确定为本实验的目标化合物。随后研究发现,IMB-DM122主要是在转录后水平通过降低：mRNA的稳定性来下调Hsc70的表达,能使Hsc70 mRNA T1/2的缩短超过78%。为了研究IMB-DM122在Hsc70 mRNA上的具体作用位点,我们将Hsc70 5’UTR及3’UTR全长分别插入到pLuc中Luc报告基因的上下游的5’端和3’端形成重组质粒,将这些质粒瞬时转染细胞后,用IMB-DM122处理,再分析含有Hsc70 3’UTR及5’UTR的Luc mRNA水平。结果显示插入3’UTR后Luc mRNA的基础表达水平下降68%,而连接5’UTR和野生型pLuc的Luc mRNA的表达水平几乎没有变化。我们通过含有HCV全长cDNA的质粒pFL-J6/JFH/JC1和Huh7.5肝细胞建立了抗HCV活病毒化合物筛选的模型,并通过该模型发现经IMB-DM122处理后的细胞,无论在mRNA水平,还是在蛋白水平,在Hsc70表达水平下调的同时,HCV也有显著下降。在转染过表达Hsc70的细胞中,相比于对照组,经IMB-DM122处理后,得到更为显著的抗HCV效果,HCV表达水平下降至未处理水平的1／16。上述结果提示,IMB-DM122的抗HCV作用是通过下调胞内Hsc70水平起作用的。进一步通过纯化病毒颗粒并分析后发现,经IMB-DM122处理后的受感染细胞所产的包装在病毒颗粒中的Hsc70含量有显著的下降,所产生的子代病毒的感染力也显著降低。这不仅验证了以往的研究结果,更重要的是开创了用化学方法在细胞内通过下调Hsc70达到抑制HCV复制的研究策略。为了评价药物作用的安全性,我们在细胞毒性实验中发现,IMB-DM122在体外对细胞毒性低；在小鼠体内实验中,腹腔注射浓度高达1000mg/kg也未见任何生长异常,给药后的血液样本中,各项肝肾功能指标也未见异常。IMB-DM122通过宿主蛋白Hsc70的下调来降低HCV颗粒的感染性,达到良好的抑制病毒复制的效果,并具有较少副作用和降低病毒发生耐药性突变的优势,该作用机制与IFN为核心的治疗策略和其他各种以病毒酶为靶点的病毒抑制剂完全不同,因此我们认为,下调Hsc70表达可能成为新的抗HCV病毒药物的机制。第二部分：人类载脂蛋白B mRNA编辑酶催化多肽样蛋白3G (human apolipoprotein B mRNA-editing enzyme catalytic polypeptide 3G protein, hA3G)属于细胞限制因子,具有一定的组织分布特异性,仅在“非允许性”细胞中表达。hA3G通过特定的包装机制在HIV-1复制过程中选择性包装进入病毒毒粒。在HIV-1的逆转录过程中,hA3G主要通过催化病毒负链cDNA中的dC脱氨为dU,在病毒基因组中引入广泛的超突变从而发挥抗病毒作用。但是HIV-1编码的Vif蛋白可以通过泛素—蛋白酶体的降解途径来拮抗APOBEC3G的抗病毒活性。本文将分别从hA3G的包装机制,hA3G在不同HIV-1宿主细胞中的表达水平,以及筛选小分子化合物抑制Vif介导的hA3G降解三个方向做探索性研究,并探讨其作为潜在抗HIV-1药物靶点的可能性。我们构建不同长度的hA3G截短质粒,分别单独包含了氨基端CDA区,锌指结构域的连接区(第104-156位氨基酸),羧基端CDA区。构建的质粒与HIV-1 Gag共转染细胞后,分别检测病毒颗粒和胞内蛋白表达,发现只有含第104-156位氨基酸的截短蛋白可以正常包装进入Gag VLP中,而该段缺失的截短蛋白,不能或只有少量包装进入Gag VLP中,该结果表明hA3G第104-156位氨基酸是hA3G包装进入病毒颗粒所必需的。通过实时荧光定量PCR相对定量检测实验室常用的HIV-1相关细胞系中内源性hA3G mRNA的表达水平,发现HIV-1宿主细胞H9、CEM、MT4都有不同水平的hA3G表达,而人肾上皮细胞293T中hA3G表达水平极低,hA3G表达水平在四种细胞中的高低顺序为：H9>CEM>MT4>293T。该结果表明人体固有免疫机制与HIV病毒的长期斗争与进化中,使hA3G的表达具有倾向于病毒易感细胞的组织分布特异性。后续实验中,我们将利用该细胞模型分析化合物的抗HIV-1活性是否通过hA3G机制。通过构建抑制Vif介导的hA3G降解的化合物筛选模型,我们从本研究所化合物库中成功筛选到活性化合物IMB-26／35,并通过Cul5 E3连接酶体系确定IMB-26／35是特异性的拮抗Vif对hA3G的降解,而非作用于蛋白酶体通路。我们将IMB-26／35及其部分衍生物通过表面等离子共振实验分析化合物与hA3G或Vif的体外结合活性和主要结合靶点。结果显示,IMB-26／35及其衍生物可与hA3G直接结合,但不与Vif结合,表明该类化合物通过与hA3G直接结合,抑制了Vif与hA3G相互作用,从而实现对hA3G抗病毒活性的保护。然后,我们利用H9、MT4和SupTl(“非允许性”细胞,低水平表达hA3G)细胞模型,检测化合物IMB-26／35的抗HIV-1活性,发现IMB-26／35的抗HIV-1活性与该三种细胞中hA3G表达水平呈正相关,分别使三种细胞中HIV-1水平下降约97%,85%和20%,验证了细胞模型的可靠性。然后用IMB-26／35及其衍生物处理经HIV-1感染的MT4细胞,检测其抗病毒活性与细胞毒性。结果显示,化合物363、2611、2613、2621等都具有较好的抗病毒效果,而351具有更低的细胞毒性。随后在小鼠体内急毒实验中,未见药物处理的小鼠死亡和显著体重改变,肝肾功能指标未见异常,并且主要器官组织均未见指标组化异常,表明化合物IMB-26以hA3G/Vif为靶点的抗HIV-1作用机制不会影响主要器官的生理功能,是相对安全可靠的。综上所述,我们的研究从分子水平到细胞水平,再到体内动物毒性实验,不仅旨在对细胞宿主蛋白为靶点的新的抗病毒机制进行理论研究,而且对新机制成为潜在抗病毒治疗方案的可能性进行探索性实践分析。综合上述两部分实验结果,我们认为该新机制具有较好的抗病毒活性,对宿主相对安全的特点,因此,与现有的病毒抑制剂联合应用可能会帮助解决病毒耐药性问题的出现。更多还原

【Abstract】 Targeting viral enzymes is an effective approach leading to potent drugs; however, newly discovered antiviral agents that act at different binding sites on the enzymes or different viral proteins generate new drug-resistant mutations. It has become very crucial to identify novel drug targets for the control of drug-resistance. This study explored the possibility of using cellular molecules as drug targets to control viral infection. The in vitro investigation has been divided into two parts, for HCV and HIV-1, respectively.Part I. It has been known that viruses interact with host cellular proteins in their replication cycle, and employ some of them for viral proliferation. Recent study has indicated host Hsc70 protein is packaged into the hepatitis C viral (HCV) particles as a structural component of the virus in the assembly process. It helps HCV RNA releasing into the cytoplasm in the next infection cycle. We investigated whether chemically down-regulating host Hsc70 expression could be a novel strategy to interrupt HCV replication. Using Hsc70 mRNA assay, compounds were screened. IMB-DM122 was found to be an effective and safe inhibitor for Hsc70 mRNA/protein expression in human hepatocytes. IMB-DM122 inhibited HCV replication through destabilizing Hsc70 mRNA, and the T1/2 of host Hsc70 mRNA was reduced by 78% after the compound treatment. The Hsc70 mRNA 3’UTR sequence is the element responsible for IMB-DM122’ s effect on Hsc70 mRNA. The compound appears to be highly efficient to the Hsc70-related HCV replication. Treatment of the HCV infected hepatocytes with IMB-DM122 reduced the virion encapsidation of Hsc70, and therefore made a break in HCV replication and infection cycle. IMB-DM122 showed a considerably good safety in vitro as well as in vivo with no indication of harmful effect on liver and kidney functions. It is our conclusion that Hsc70 might be a new drug target and mechanism to inhibit HCV proliferation.Part II. On the other hand, host innate immunity system fights against viral intruders in a variety of strategies. Human APOBEC3G (hA3G) is part of this system of host cell and has cytidine deaminase activity. It specifically incorporates into the virion during HIV-1 replication. In the HIV-1 reverse transcription process, hA3G deaminates dC to dU in the first minus strand cDNA, and then induce extensive hypermutation in the viral genome. Unfortunately, HIV-1 Vif counteracts the activity of hA3G by an ubiquitin-proteasome-mediated degradation of hA3G, suggesting that the interaction between Vif and hA3G might be a potential target for novel anti-HIV-1 drugs. In this part of work, we first established a screening system to discover compounds that protect hA3G from Vif-mediated degradation. After screening over 8634 samples, two active compounds, IMB-26 and IMB-35, were identified to be blockers for the Vif/hA3G interaction. The compounds specifically inhibited the degradation of hA3G by Vif without interference of the proteasomal function, and efficiently inhibit HIV-1 replication in hA3G-containing cells but not those null of hA3G The anti-HIV activity of IMB-26/35 positively correlated with the endogenous hA3G level in host cells. BIAcore examination found that IMB-26/35 protected hA3G by inhibiting the interaction between Vif and hA3G, and the hA3G protein appeared to be the receptor molecule for binding of the compound. The compounds were safe with a therapeutic index over 200 in vitro. LD50 of IMB-26 in mice was over 1000 mg/kg (ip) with no toxicity in liver and kidney. These data together indicate that Vif/hA3G interaction a new anti-HIV target, and IMB-26/35 potential leads for the discovery of new mechanism anti-HIV drugs.In summary, host cellular proteins that relate to viral replication could be novel drug targets to inhibit viruses, if their intracellular concentration is not a key factor for cell survival. It might be a potential strategy against drug resistance.更多还原