【作者】 刘然；

【导师】 秦鄂德； 秦成峰；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2010， 博士

【摘要】 登革病毒(dengue virus, DV)是重要的虫媒黄病毒(Flavivirus),能引起人类的登革热和登革出血热。目前尚无有效的登革疫苗和抗病毒药物。登革病毒基因组为一条单股正链RNA分子,可作为信使RNA直接起始翻译,并通过其中仅有的一条长读码框(open reading frame, ORF)编码病毒所有的十种特异蛋白。病毒基因组RNA(genomic RNA, gRNA)亦可作为模板指导子代gRNA的合成,该过程遵循"gRNA——gRNA互补负链——子代gRNA"的模式。由此可见,登革病毒基因组的表达和复制都围绕着一条gRNA分子进行。因而通常认为登革病毒并不产生亚基因组RNA分子(sub-genomic RNA, sgRNA)。登革病毒基因组的表达和复制受一系列基因组顺式作用元件(cis-acting element)和反式激活因子(trans-activator)的调控。登革病毒的顺式调控元件主要位于gRNA 5’和3’端上的非编码区(non-coding region),其中包含了多个高度保守的RNA二级结构。这些RNA结构元件能够特异性地识别病毒复制的反式激活因子,从而对病毒gRNA的翻译和复制过程进行调控。根据病毒基因组结构特征,通常认为,5’NCR的功能与gRNA的翻译有关。而3’NCR负责调控gRNA的复制。通过前期的研究,我们发现3’NCR中还存在翻译增强元件(Translation enhancement element, TE)。囿于现有的登革病毒复制模型,还不能完满地解释上述矛盾,推测在病毒基因组的复制过程中还存在其它调控机制。从进化上看,单正链RNA病毒的基因组结构特征与其采取的基因组复制调控策略存在密切的关系。值得注意是,一些单正链的RNA病毒虽然在基因组结构上与登革病毒近似,但在复制上还需要产生源于病毒基因组3’末端的亚基因组RNA分子(3’sub-genomic RNA,3’sgRNA)。利用这类3’sgRNA分子,病毒既可增强基因组3’末端基因的表达,或进一步对自身复制进行调控。但对登革病毒,目前尚无相关的研究。因此,倘若登革病毒在复制过程中确实存在类似的3’sgRNA分子,这无疑对阐明登革病毒复制调控策略具有重要的意义,并为研究登革病毒复制的分子机制提供新的思路。为此,本研究通过Northern杂交方法对登革病毒感染的细胞和组织总RNA进行了鉴定。首先根据登革四个血清型病毒基因组3’NCR序列比对结果,选定基因组3’最末端的高度保守序列作为杂交探针的靶点,利用体外转录方法制备获得地高辛标记的探针RNA分子。初步的杂交结果证实登革病毒在复制过程中确能产生一类源于基因组3’末端的亚基因组RNA分子(3’sgRNA)。进一步的杂交结果表明,从登革四个血清型病毒所感染的BHK-21细胞与乳鼠脑组织中均可检出该类3’sgRNA分子,表明它的产生是登革病毒复制中的普遍现象。同时还观察到登革2型中的一株病毒(DV2-43)能同时产生三条3’sgRNA分子的株特异性现象。在病毒感染后的不同时间点上,随着病毒gRNA复制增多,3’sgRNA在宿主细胞中也不断累积。这一现象提示3’sgRNA的产生很可能与病毒复制有关。此外,在不同组织来源的宿主细胞中,病毒3’sgRNA的生成量存在明显的差异。这提示了3’sgRNA的产生还与宿主因素存在某种关联。上述结果为阐明登革病毒3’sgRNA的产生机制和潜在的生物学功能提供了重要的线索。在此基础上,为明确登革病毒3’sgRNA产生的缘由,本研究利用5’RACE法获得了登革病毒3’sgRNA的序列信息。比对结果显示登革病毒3’sgRNA的5’端起始于病毒基因组3’NCR的内部,这表明3’sgRNA实质上是游离形式的3’NCR分子。3’sgRNA的5’起始序列高度保守,且能形成一个稳定的RNA茎环结构SL,提示该段序列可能具有启动子样功能。RNA结构预测还表明,保守序列与SL结构的茎部序列恰好吻合,说明3’sgRNA的产生与SL结构的形成有关。在细胞5’核酸外切酶XRN-1的作用下,SL结构的存在可使病毒gRNA及其模拟物VIRG分子经部分降解产生与3’sgRNA类似的RNA片段。另一方面,Western blotting结果表明,XRN-1可被BHK-21等真核细胞表达。上述结果初步表明XRN-1参与了登革病毒3’sgRNA在细胞内的生成。我们认为3’sgRNA的产生是RNA结构与宿主核酸酶类相互作用的结果,这为从分子机制上干扰3’sgRNA的产生提供了可能的靶点,并为揭示该分子的生物学功能奠定了基础。为进一步探明3’sgRNA的生物学功能,本研究通过将3’sgRNA转染细胞的方法,对3’sgRNA在病毒基因组翻译和复制中的作用进行的鉴定。我们首先利用病毒小基因组VIRG对登革病毒gRNA的翻译起始过程进行了模拟,从中观察到3’sgRNA的转染能下调VIRG报告基因的表达量。而对病毒基因组RNA的定量分析结果表明,预先转染3μg的3’sgRNA转录体,可使105个细胞中的病毒基因组RNA拷贝数上升一个数量级,并且3’sgRNA转染量与其对复制的增强作用间存在依赖性关系。登革病毒在3’sgRNA转染的细胞上能形成更多的病毒蚀斑,说明3’sgRNA不仅对gRNA翻译和复制起调节作用,它的存在也有利于病毒的增殖。为明确3’sgRNA的作用机制,本研究对3’sgRNA包含的基因组环化序列进行了突变,以求破坏3’sgRNA与病毒基因组5’端的互补结合。结果表明,突变可削弱3’sgRNA对病毒复制的增强作用,这说明3’sgRNA的调节作用依赖环化序列介导的RNA-RNA相互作用。由此我们认为登革病毒3’sgRNA是一种通过反式作用对病毒复制进行调节的非编码RNA分子。本研究首次发现登革病毒在复制中能产生一类非编码的3’sgRNA分子,并表明3’sgRNA的产生与其5’端的保守的茎环结构有密切关系,同时初步揭示3’sgRNA的产生还需要一种宿主的核酸酶类参与。在此基础上,证明了3’sgRNA分子具有调节病毒gRNA翻译和复制的功能,即说明登革病毒基因组3’NCR还可通过反式作用调控病毒的复制。上述发现为深入阐明登革病毒复制的分子机制奠定了基础。更多还原

【Abstract】 Dengue virus was an important member of flavivirudae which cause dengue fever and hemorhagic fever. Until now dengue vaccines and antiviral drugs were unavailable.Viral genome is a single strand positive RNA molecule, which could serve as viral messenger RNA to initiate translation. All of ten viral proteins were translated together from a single open-reading frame contained in viral genomic RNA(gRNA), then spliced apart by host signalase and viral proteases. In replication, viral gRNA also direct synthesis of progeny gRNAs as template. According to genomic structure and strategy of gene expression, it is believed that none of sub-genomic RNAs were involved in dengue viral replication, except gRNA positive strand and its complementary negative RNA.Translation and replication of dengue viral gRNA were under the control of series genomic cis-acting elements and trans-activators. Most of cis-acting elements were located in genomic 5’and 3’terminal non-coding region, which could formed several highly conserved RNA structures. These structured RNA elements exserted regulation and control function to viral replication by specific binding to trans-activators. According to viral gRNA’s organization, it was believed that 5’NCR and 3’NCR played their roles in translation and replication, respectively. several studies had indicated that 3’NCR could also recruite eukaryotic translation initiation factors. Previously, we found translation enhancement elements were located in 3’NCR. Current replication models of dengue virus could not explaine this discrimination. We proposed that other regulation stradegy were empolied by dengue virues.In the perspective of RNA viruse evolution, organization of viral gRNA partly determined the replication strategy which virus used. We were inspired by some plant positive single-strand RNA viruses, which had similar genomic organization as dengue viruses did, but also could generate viral sgRNA which was specifically associated with gRNA 3’terminus. Intrestingly, this viral RNA species played distinct roles in viral life cycle. For dengue viral and other animal RNA viruses, it was still a question. If such 3’sgRNA did exist, it might help us to have a better understanding on viral replication. Furthermore, its potential functions would offer novel ideas for study on viral replication mechanism.To indentify the existence of dengue viral 3’sgRNA, total RNA derived from virus infected cells and tissue were analyized by northern blotting. According to alignment results of 3’terminal sequence derived from four types viruses, an extremely conserved region was chosen to be targets of sepecific probes. RNA probes were labelled by digoxin during in vitro transcription. Northern blotting analysis showed that dengue viruses did generated viral 3’sgRNA during replication. Further experiments confirmed that all of four types of dengue viruses could produce similar 3’sgRNAs both in infected BHK-21 cells and suckling mouse brain tissue, which suggested that generation of 3’sgRNA was a ubiquitous event during viral replication. Unexpectedly, we also witnessed a unique strain of DV2 produces three species of sgRNAs simutaneously in comparison with other isolates. Furthermore, we noticed that 3’sgRNA accumulated in BHK-21 cells along with viral gRNA synthesis, which had strongly indicated that 3’sgRNA properly play roles in viral replication. On the other hand, the quantity of 3’sgRNA produced in distinct host cells were distinct. It had implied that host cell factors also contribute to the rise of 3’sgRNA.To clarify the property of dengue viral 3’sgRNA, its sequence information of 3’sgRNA were obtained by 5’RACE, then sequnences alignment result had showed that the initiation site of 3’sgRNA was all located in 3’non-coding region. It showed that 3’sgRNA was a free 3’NCR fragment per se. Alignment work also revealed an conserved region appeared at the 5’initiation site of 3’sgRNA, which indicated that this sequence could be a promoter-like element. Secondary structure predication further revealed that a RNA stem-loop structures emergered at the iniation site, which suggested that RNA structure might have impact on 3’sgRNA generation. Then ribonuclease degradation experiment was carried out to further tesitify the biochemical propertity of SL structure. It was showed that SL was invuneralbe to degradation of a enkaryotic 5’exo-ribonuclease XRN-1. Both viral full length RNA clone and mini artificial gRNA contained a reporeter gene could generate 3’sgRNA analogs in vitro by XRN-1.On the other hand, XRN-1 was detected in BHK-21 cells, although its expression was at a low level. Taken together, our study proposed a potential mechasim of 3’sgRNA biogenesis, in which host ribonucleases and a stem-loop structure took part in 3’sgRNA generation.Our previous study had showed that 3’sgRNA contained all of cis RNA elements in 3’NCR. Accumulation of 3’sgRNA was also observed. Taken them together, we assumed that 3’sgRNA stand a great possibility to exert its function to viral replication. By 3’sgRNA transfecting, we elevated the quantitiy of 3’sgRNA in host cells. Firstly, we constructed a viral mini gRNA, which contained viral intact NCRs and a reporter gene in order to mimic viral gRNA translation. Then we found 3’sgRNA could suppress reporter gene expression. To investigate 3’sgRNA function in viral gRNA synthesis,3μg 3’sgRNA was transfected into host cells, then copy numbers of viral gRNAs were found increased about 10 times in 105 transfected cells in comparison to untransfected cells. Further observation revealed this replication augument was dependent on transfection dose. Consitently, plague formed by progeny virus particles were also increased. It suggested that 3’sgRNA not only regulate viral gRNA replication and translation, also facilitate viral propergation. To confirm the mechanism of 3’sgRNA maniplutaing viral replication, a pair of cyclization sequences was mutant to depelete RNA-RNA interaction between viral genome 5’and 3’terminus. Mutation partly reduced replication enhancement mediated by 3’sgRNA. We proposed that new discovered 3’sgRNA was a trans-acting ribo-regluatior in viral replication.In our study, a novel 3’sub-genomic RNAs of dengue virues was indentified and characterized for the first time. Sequencing revealed that 3’sgRNA were small non-coding RNA derived from 3’NCR. Structural analysis indicated its interaction with host cell exoribonuclase, by which could give the rise of 3’sgRNA and this mechanism appeared to be a novel pathway in RNA viruses for generating sgRNA. Finally, it showed that 3’sgRNA had influence on both viral gRNA synthesis and translation. We proposed that 3’sgRNA was a trans-acting ribo-regluatior in viral replication. Our study updated our understanding of dengue viral replication, and also shed light on viral replication and host cell interaction.更多还原