【作者】 李晓峰；

【导师】 秦鄂德；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2008， 博士

【摘要】 西尼罗病毒是黄病毒科黄病毒属的重要成员。其基因组为单股正链RNA分子,包含一个单一读码框,分别编码3个结构蛋白和7个非结构蛋白(NS1、NS2a、NS2b、NS3、NS4a、NS4b和NS5)。基因组5′和3′端为非编码区(untranslated region,UTR),含多个保守序列和二级结构,维持这些保守序列间的互补以及二级结构的稳定是病毒RNA复制的关键。非结构蛋白NS3和NS5是病毒RNA复制复合体的重要成分,因此也是病毒基因组复制的关键蛋白。对UTR的功能研究多集中于3′端,采取的策略多为将突变引入感染性全长cDNA克隆后观察恢复病毒生物学性质的变化。对NS3和NS5蛋白的研究也集中于其酶学性质,而二者与病毒基因组非编码区的相互作用研究甚少。关于西尼罗病毒基因组5′-UTR与NS3和NS5蛋白的相互作用目前尚未见报道。因此,对西尼罗病毒5′-UTR中的保守序列和二级结构如何与病毒和细胞蛋白相互作用,这种相互作用的生物学效应及其时间与空间的转换机制进行研究,可为该病毒复制的分子机制提供新的理论依据。为此,本研究首先构建并鉴定了西尼罗病毒Chin-01(01)株基因组感染性全长cDNA克隆。之后经原核表达和纯化后获得了具有酶活性的NS3、NS5蛋白RdRp活性区(NS5pol)及完整的NS5蛋白(NS5F)。进一步对包括Chin-01株在内的多株西尼罗病毒的5′-UTR进行了生物信息学分析,并初步确定突变靶点;观察了NS5pol和NS5F以突变的重组亚基因组为底物时的复制能力及与底物的结合能力;同时还观察了NS3对NS5与突变重组亚基因组结合能力和复制能力的影响;再将由此确定的与NS3和NS5相关的复制元件的突变位点引入基因组全长感染性cDNA克隆,观察突变恢复病毒生物学性质的变化,并提出了西尼罗病毒基因组子代RNA合成起始时可能的复制复合体模型。这也为西尼罗病毒乃至其他黄病毒基因组复制的分子机制提供了理论依据。1.西尼罗病毒01株基因组感染性全长cDNA克隆的构建与鉴定首先,构建了Chin-01株基因组感染性全长cDNA克隆。将病毒基因组分为5段进行RT-PCR扩增后获得片段F1～F5,其中在F1片段所含的基因组5′端上游引入了SP6启动子识别序列。进一步将F1～F3及F4和F5分别连接,构建了Chin-01株的5′和3′半分子。为了获得与病毒基因组一致的3′末端,通过PCR去除基因组原有的XbaI识别序列,并在原有3′半分子的3′末端引入该酶的识别序列。最后将5′和3′半分子连入pBluescriptⅡKS载体,构建了3′末端分别为NotI和XbaI识别序列的重组质粒pBS-F-N和pBS-F-X,进一步的酶切鉴定及接头处序列测定的结果表明,成功获得了Chin-01株基因组全长cDNA克隆。为了观察由全长cDNA克隆所得的RNA是否具有感染性,将体外转录制备的RNA转染BHK-21细胞后获得恢复病毒BS-F-N和BS-F-X。对恢复病毒基因组的RT-PCR的鉴定结果显示,恢复病毒的基因组序列为野生病毒的特异序列,并含有引入的酶切位点;间接免疫荧光的结果表明恢复病毒能够在敏感细胞中表达自身特异蛋白;恢复病毒感染细胞后也出现了与野生病毒相似的细胞病变,但出现病变的时间比野生病毒延迟12～16 h。在Vero细胞上也能形成与野生病毒形态相似的空斑,但直径略小于后者,出斑时间也延迟15～20 h。一步生长曲线的结果显示,恢复病毒的滴度在感染细胞72 h后达最高,为10~5,而野生病毒的滴度在48 h后即达峰值,为10~6,表明恢复病毒与野生型病毒具有相似的感染性和增殖特性。此外,无论在自身蛋白表达水甲、空斑形成时间还是病毒的增殖能力,恢复病毒BS-F-N均弱于BS-F-X。而测序结果显示,BS-F-N的3′端仍有经NotI酶切后形成的非病毒核苷酸。由此推测二者之间复制与增殖能力的差异可能是3′末端的若干非病毒核苷酸的影响所致。以上结果表明,成功获得了西尼罗病毒01株基因组的两株感染性全长cDNA克隆(pBS-F-N和pBS-F-X),最终选取与野生病毒感染性更接近的pBS-F-X作为进一步构建复制相关元件突变体的骨架。2.西尼罗病毒01株NS3、NS5pol和NS5蛋白的表达纯化及酶活性的鉴定获得具有完整功能活性的NS3和NS5蛋白是研究西尼罗病毒蛋白与基因组相互作用的前提。为此,首先将PCR扩增所得的NS3、NS5pol和NS5F的编码序列连入原核表达载体pET-28a后获得相应的重组表达质粒,并在15℃,IPTG为0.2 mM条件下进行诱导表达。分别用抗6His单抗和西尼罗病毒免疫小鼠后的腹水多抗对各可溶性目的蛋白进行Western blot检测,结果表明表达的蛋白为病毒特异蛋白。进一步经Ni柱亲和层析后获得纯度在90%以上的重组NS3、NS5pol和NS5F蛋白。体外RNA解旋酶活性测定的结果表明NS3蛋白具有RNA解旋酶活性。体外RdRp活性测定和凝胶阻滞实验的结果表明,NS5pol和NS5F具有完整的RdRp活性,而且该活性具有模板和二级结构的特异性。获得的具有RdRp活性的NS5pol和NS5F以及具有RNA解旋酶活性的NS3蛋白,也为下一步复制相关蛋白与病毒基因组5′-UTR的相互作用研究奠定了基础。3.病毒基因组5′端与病毒复制相关蛋白的相互作用为了筛选出5′-UTR二级结构的关键核苷酸作为突变靶点,首先对多株西尼罗病毒5′-UTR的序列进行了比对,发现该区可分为A′、B′和C′亚区。选取三个亚区中的若干位点作为突变靶点,通过缺失突变和定点诱变构建了10个仅含5′-UTR,且各亚区呈不同结构的突变体。考虑到3′-UTR可能影响基因组与蛋白的相互作用,进一步构建了对应的10个5′-UTR突变的,且包含完整UTR的突变重组亚基因组。通过体外RdRp活性测定及凝胶阻滞实验观察NS5pol和NS5F以上述突变体为底物时的RdRp活性及与底物的结合能力。结果显示,无论以何种突变体为模板,5′-UTR第28、37、38和43位核苷酸同时突变及第46～60位的缺失均导致NS5无法合成子代RNA。以突变的5′-UTR为模板时,只有第8和9位突变才导致NS5既无法合成子代RNA也无法与模板结合。而以突变的完整UTR为模板时,只有第8、9、69和70位同时突变才导致NS5丧失合成子链的能力和模板结合的能力。上述结果表明,西尼罗病毒5′-UTR中C′区的第28、37、38和43位及B′区第45～60位核苷酸可能是NS5发挥其RdRp活性的关键位点;而A′区的第8、9、69和70位核苷酸可能与3′-UTR一同在RNA复制中发挥作用。此外还发现,5′-UTR中的不同突变位点也可影响NS5在合成子代RNA时采取的起始方式。进一步观察了NS3蛋白对NS5pol和NS5F RdRp活性及与模板结合能力的影响。结果显示,加入NS3后,无论以何种突变体为底物,缺失第46～60位核苷酸后NS5即无法发挥其RdRp活性,而且NS5pol的RdRp活性及与模板的结合能力与未加NS3时一致。以突变的5′-UTR为模板时,NS5F的酶活性和结合能力与未加NS3时一致;而以突变的完整UTR为模板时,第28、37、38和43位同时突变后,NS5F仍能合成子代RNA,而且合成子代RNA的量随NS3的量的增加和与之孵育时间的延长而增加;而第8和9位突变时,NS5F仍无法合成子代RNA;只有与69和70位同时突变后,NS5F才能合成微量的RNA,并与模板特异性结合。此外,NS3还可与3′-UTR特异性结合。由此推测,NS3可能借助3′-UTR来促进NS5酶活性及与模板的结合。NS3存在时,NS5聚合酶活性区与完整的NS5的RdRp活性及与模板结合能力的差异也表明,NS5蛋白N端未包含NLS序列的非RdRp区可能含有与NS3相互作用的位点。综合上述结果表明,西尼罗病毒5′-UTR的B′区第46～60位核苷酸可能是不依赖于NS3的,且与NS5行使其RdRp活性密切相关的位点;而C′区的第28、37、38和43位以及A′区中由第8、9、69和70位核苷酸所维持的茎结构可能含有NS3和NS5协同作用的位点。同时,NS3可能借助3′-UTR或与NS5蛋白N端未包含NLS序列的非RdRp区的结合来发挥作用。4.病毒基因组5′端复制相关元件突变的恢复病毒的生物学性质为了进一步验证体外实验所得结果,将A′区的第8、9、69和70位和C′区第28、37、38和43位的点突变及B′区第46～60位的缺失突变引入Chin-01株基因组感染性全长cDNA克隆pBS-F-X,还引入B′和C′间的第20～45位的缺失突变,以便进一步观察该区结构对病毒复制的影响。将突变的全长cDNA克隆经体外转录后转染敏感细胞,通过间接免疫荧光、空斑试验、一步生长曲线及基因组的RT-PCR鉴定,对获得的恢复病毒的生物学性质进行观察。结果显示,B′区第46～60位的缺失及A′区中由第8、9、69和70位维持的茎结构的破坏均导致恢复病毒的复制能力显著降低,而B′和C′间的第20～45位的缺失及C′区第28、37、38和43位的突变对病毒的增殖能力并无明显影响。以上结果表明,西尼罗病毒5′-UTR的A′区的茎结构(第8、9、69和70位)和B′区的侧环(第46～60位)是病毒维持其复制的关键位点。而C′区结构(第20～45位)对病毒复制的影响较小。本研究的结果表明,WNV基因组5′-UTR的A′区茎结构及B′区侧环是病毒复制的关键位点;而NS3也可与3′-UTR特异性结合,并可以促进NS5的RdRp活性及与突变体亚基因组的结合。综合上述结果,我们提出WNV基因组子链RNA合成起始阶段可能的复制复合体的组成模型,即NS5首先识别5′-UTR,进一步结合NS3蛋白,或通过其他因子的介导来结合NS3,通过后者与3′-UTR的结合来拉近NS5与3′末端的距离,从而利于子链的合成的起始。更多还原

【Abstract】 West Nile virus(WNV)belongs to genus Flavivirus within the the family Flaviviridae.WNV possesses a single-stranded,positive-sense RNA genome of approximately 11 kb,which contains a long open reading frame coding for a polyprotein precursor.The polyprotein is processed into three mature structural proteins(C,prM,and E)and seven nonstructural proteins(NS1,NS2A,NS2B,NS3, NS4A,NS4B,and NS5)by cellular and viral proteases.Flanking the genome were 5’ and 3’untranslated regions(UTR)containing conserved sequences and secondary structures which appear to be essential for viral RNA replication.NS3 and NS5, which are thought to be the main structural components of RNA replication complex,are the key proteins responsible for the viral RNA replication.The function of 3’-UTR has been characterized in great detail,the common strategy used in the study of which is to observe the biological consequences of mutations introduced into the infectious full-length cDNA clone.While the enzymatic functions of NS3 and NS5 have been well characterized,the interactions of NS3 and NS5 with UTR of genome remain unclear.In addition,there still have been no reports on interactions of NS3 and NS5 with WNV 5’-UTR.Therefore,study of functions of interactions of viral and cellular proteins with conserved sequences and secondary structures in 5’-UTR of WNV genome will help to clarify molecular details of the viral replication.In this study,firstly an infectious full-length cDNA clone of WNV Chin-01 strain was constructed.Then full-length NS3,RNA-dependent RNA polymerase (RdRp)domain of NS5(hereafter named NS5pol)and complete NS5(hereafter named NS5F)with an N-terminal histidine tag were expressed in E.coli and purified, respectively,which were enzymatically active.Further a bioinformatic analysis of 5’-UTRs of several WNV strains including strain Chin-01 was performed and several nucleotides were selected as mutations introduced into the 5’-UTR of WNV.Then RdRp activity of NS5pol and NS5 were observed using different mutant 5’-UTRs as templates,which was followed by the observation of the effects of NS3 on the polymerase activity of NS5 using mutant 5’-UTRs and capability of binding of NS5 to mutant templates.Finally,the mutations in elements related to NS3 and NS5 in 5’-UTR involved in viral replication were introduced into the infectious full-length cDNA clone of Chin-01 strain.Then the biological characteristics of the recovered virus were observed.A model for replication complex formed in the stage of initiation of negative-strand synthesis was proposed.The results in this study may shed light on molecular mechanisms of genome replication of WNV and other flaviviruses.1.Construction and identification of two infectious full-length cDNA clones of WNV Chin-01 strainFirstly,a full-length cDNA clone of Chin-01 strain was constructed.Five cDNA fragments(F1～F5)were synthesized from genomic RNA through RT-PCR to cover the complete WNV genome.Then F1～F3 and F4～F5 were cloned together into plasmids to yield 5’ and 3’-halves of genome of Chin-01 strain,respectively.In order to obtain RNA possessing authentic 3’-end,two XbaI sites in WNV gemome were abolished and a unique XbaI site was introduced into the 3’-end of the 3’-half of genome of Chin-01 strain by site-directed PCR mutagenesis.The 5’ and 3’-halves of genome were assembled into plasmid pBluescriptⅡKS to form the full-length cDNA clone of WNV(pBS-F-N and pBS-F-X).The complete WNV cDNA is positioned under the control of SP6 promoter elements for in vitro transcription.The virus-specific sequence of each intermediate cloning product and junction sequences were validated by sequence analysis and restriction digestion.In order to observe the infectivity of RNA from the full-length cDNA clones, Capped RNA transcript synthesized from the linearized full-length cDNA plasmid by using SP6 RNA polymerase was electroporated into BHK-21 cells and recovered virus BS-F-N and BS-F-X were obtained.The fragments spanning the genetic markers were amplified through RT-PCR from RNA extracted from either parental or recovered viruses and validated by sequence analysis,the results of which clearly showed that virus recovered from the transfected cells was derived from the infectious full-length RNA;The results of IFA used to detect viral protein expression in BHK-21 cells transfected with WNV RNA transcript also indicated that specific viral proteins were expressed by progeny virus.No qualitative differences in CPE were observed between parental and recovered viruses,but the CPE from recovered virus showed after a delay of approximately 12～16 h.Furthermore,there was no difference morphology on Vero cells between the recombinant and parental virus,but the size of plaques of recovered virus,which appeared after a delay of approximately 15～20 h, was marginally smaller in diameter than that of parental virus.One-step growth curves were similar for both recombinant and parental viruses on BHK-21,the peak titers of which were 10~5 PFU/ml at 72 h p.i and 10~6 PFU/ml at 48 h p.i,respectively. These data suggest that the parental and recovered viruses are indistinguishable in replication and spread in mammalian cells.In addition,the levels of viral protein expression and RNA replication of recovered virus BS-F-N whose 3’-end contained several nucleiotides unrelated to virus were lower than that of BS-F-X.This disparity is likely due to the nucleiotides unrelated to virus postioned at 3’-end of BS-F-N.The results above suggested two infectious full-length cDNA clones of WNV Chin-01 strain were obtained successfully(pBS-F-N and pBS-F-X).The pBS-F-X whose infectivity was similar to parental virus was used to construct the mutant in further.2.Expression and purification of enzymatically activive of WNV Chin-01 strain NS3,NS5pol and NS5FIt is crucial for study of interactions between viral proteins and genome of WNV to obtain NS3 and NS5 possessing enzymatic activity.In this regard,firstly the PCR-fragments encoding NS3,NS5pol and NS5F were cloned into prokaryotic expression plasmid pET-28a resulting in the corresponding recombinant expression plasmids. Expressions for all proteins were carried out at 15℃after induction with 200μM IPTG.Recombinant proteins were identified after SDS-PAGE by Western blotting using anti-6His monoclonal antibody and polyclonal rabbit antiserum against the WNV,respectively.The purified recombinant proteins NS3,NS5pol and NS5F with purity above 90%were obtained through affinity chromatography on a Ni-column. The results of RNA helicase assay in vitro indicated that recombinant protein NS3 possessed the intact helicase activity;In vitro RdRp assay and EMSA undertaken further showed that the recombinant NS5pol and NS5 exhibited high RdRp activity, and that the RdRp activity was dependent on the sequence and secondary structure of RNA.The recombinant fusion NS3,NS5pol and NS5F of WNV obtained layed a foundation to identify elements in WNV genome essential for viral RNA synthesis. 3.Interactions between viral proteins related to viral replication and 5’-UTR of WNV Chin-01 strain.In order to screen the key nucleotides in 5’-UTR as targets,the secondary structures of 5’-UTRs of several strains of WNV firstly were predicted firsly,the results of which showed that 5’-UTR consists of subdomain A’,B’ and C’.Then mutations were introduced into several positions selected as targets in these subdomains by PCR mutagenesis,and ten mutants containing 5’-UTR,secondary structures of subdomains of which are different from each other,were constructed. Given to the effect of 3’-UTR on the interactions of viral proteins with viral genome, the corresponding ten mutants containing intact UTR were constructed further.Then RdRp activity and capability of binding to templates of NS5pol and NS5F were evaluated through RdRp assay in vitro and EMSA respectively using the mutant 5’-UTRs mentioned above.In these experiments,mutations at nucleotides 28,37,38 and 43 simultaneously and deletion of nucleotides 46～60 all resulted in deficiency of RdRp activity of NS5pol and NS5F;but mutations at nucleotides 8 and 9 resulted in deficiency of RdRp activity and capability of binding to templates of two proteins using mutants containing 5’-UTR as templates;However,Only mutations at nucleotides 8,9,69 and 70 simultaneously resulted in deficiency of RdRp activity and capability of binding to templates using mutants containing intact UTRs as templates. These results indicated that the RdRp activity of NS5 might be dependent on the nucleotides 28,37,38 and 43 in subdomain C’ and nucleotides 45～60 in subdomain B’ in 5’-UTR,while nucleotides 8,9,69 and 70 in subdomain A’ might play a key role in RNA replication through interaction with 3’-UTR.In addition,mutatations in 5’-UTR could affect the mechanisms used in initiation of RNA synthesis of NS5.And then the effect of NS3 on the polymerase activity of NS5 using mutant 5’-UTRs and capability of binding of NS5 to mutant templates were evaluated.After addition of NS3,the template activity of mutant with deletion of nucleotide 46～60 was still almost abolished.But RNA synthesis was restored using NS5F and mutants containing intact UTR with mutations at nucleotides 28,37,38 and 43 simultaneously, and that the amount of RNA synthesized was increased with increasing concentrations and prolonged incubation of NS3.In addition,the mutant containing intact UTR with mutations at nucleotides 8,9,69 and 70 simultaneously was active for RNA synthesis using NS5F.Besides,NS3 had a capability of binding to 3’-UTR specifically.The results above indicated that NS3 might be able to promote the RdRp activity and improve the capability of binding to template of NS5 by interactions with 3’-UTR and (or)the N-terminal region of non-RdRp without NLS ofNS5.Altogether,these results suggested that nucleotides 45～60 in subdomain B’ of 5’-UTR of WNV might be NS3-independent and closely related to RdRp activity of NS5,While nucleotides 28,37,38 and 43 in subdomain C’ and stem-loop kept by nucleotides 8,9,69 and 70 in subdomain A’ were likely to contain the sites interact with NS3 and NS5.In addition,NS3 might play its role through interaction with 3’-UTR and the N-terminal region of non-RdRp without NLS ofNS5. 4.Biological characteristics of recovered virus with mutations in 5’-UTRIn order to verify the results obtained in vitro,mutations at nucleotides 8,9,69 and 70 in subdomain A’,28,37,38 and 43 in subdomain C’ and deletion of nucleotides 45～60 in subdomain B’ were introduced into the correspondent region of infectious full-length cDNA clone of pBS-F-X.In addition,deletion of nucleotides 20～45 in subdomain C’ was also introduced so that the effects of subdomain C’ on RNA replication could be evaluated.RNA transcript synthesized from the linearized full-length cDNA plasmids was electroporated into BHK-21 cells.Then the biological characteristics of mutant recovered viruses obtained were observed by IFA,plaque assay,one-step-growth and RT-PCR of genome.When the region of nucleotides 45～60 in subdomain B’ was deleted and the stem-loop in subdomain A’ was destroyed, The mutant recovered viruses were almost completely replication defective,but the deletion of nucleotides 20～45 and mutations 28,37,38 and 43 in subdomain C’ had no effect on viral replication.These results indicated that stem-loop and side-loop in subdomain A’ and B’ respectively may play an essential role in viral RNA replication.In this study,the results above suggested that stem-loop and side-loop in subdomain A’ and B’ respectively were critical for WNV Chin-01 strain replication; NS3 had a capability of binding to 3’-UTR specifically,and might be able to promote the RdRp activity and improve the capability of binding to template of NS5.Based on these results,A possible model for RNA replication complex formed in the stage of initiation of negative-strand synthesis was proposed,in which NS5 firstly recognizes 5’-UTR,and then binds to NS3 directly or through other factors.In virtue of the interaction of NS3 with 3’-UTR and(or)long-range RNA-RNA interactions between 5’ and 3’-UTR.the 5’ and 3’-end of the genome are brought together to facilitate RNA synthesis.更多还原