登革病毒C基因RNA二级结构及编码蛋白在病毒复制中的作用
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摘要
登革病毒(DEN)为黄病毒科(flaviviridae)黄病毒属(flavivirus)的重要成员,包括4个血清型(登革病毒1-4型,DEN 1-4)。登革病毒感染可导致登革热(dengue fever,DF)和症状更加严重的登革出血热/登革休克综合征(dengue hemorrhagic fever/dengue shock syndrome,DHF/DSS)。登革病毒基因组为长约11,000nt的单股正链RNA,包括5′非编码区(untranslated region,UTR)、单一开放读码框(open reading frame, ORF)和3′非编码区。其ORF编码3种结构蛋白(C, prM和E)和至少7种非结构蛋白(NS1,NS2A,NS2B,NS3,NS4A,NS4B和NS5)。
黄病毒的衣壳(capsid,C)蛋白是病毒核衣壳的基本构成成分,多拷贝的C蛋白将病毒RNA包裹形成核衣壳结构。黄病毒C蛋白具有4个α螺旋结构(α1-α4),且该蛋白具有功能多样性。目前已经发现部分黄病毒的C蛋白对病毒RNA复制及蛋白翻译具有调节作用,并且C蛋白对病毒复制的调节与其核定位现象有关。登革病毒C蛋白的核定位现象提示我们,在登革病毒RNA复制过程中,C蛋白也可能发挥了一定功能。除C蛋白本身外,C基因在病毒RNA复制和翻译过程中也发挥了重要作用。黄病毒C基因紧邻与病毒复制密切相关的5′非编码区,而且在C基因中已经发现了多个在病毒复制和翻译过程中发挥重要作用的RNA结构元件。位于C基因内的5′环化序列(cyclization sequence, CS)和cHP元件对于病毒的复制和翻译均具有重要意义。但目前黄病毒C蛋白及C基因中的结构元件调节病毒复制的机制还不十分清楚。在登革病毒中,尚无C蛋白参与调节病毒复制的报道,而在C基因中是否存在其它影响病毒复制的RNA结构元件的问题也没有明确的答案。
针对上述问题,本研究利用病毒复制子技术分析登革病毒C基因及其编码蛋白突变对病毒RNA复制的影响,并通过与生物信息学手段相结合对C基因中的保守RNA二级结构在病毒RNA复制过程中的作用进行分析;在发现C蛋白核定位序列与病毒RNA复制有关的基础上,利用酵母双杂交技术筛选与C蛋白存在相互作用的病毒及宿主蛋白,从而对C蛋白参与调节病毒复制的作用机制进行探讨。研究内容主要包括以下三个部分。
一、登革病毒C蛋白基因突变对病毒RNA复制的影响
为分析登革病毒C蛋白中是否存在影响病毒复制的结构,并分析其基因中是否存在除5′CS和cHP外其它影响病毒复制的结构元件,我们构建了含有不同α螺旋编码序列缺失以及核定位序列突变的C基因的登革4型病毒复制子。然后通过测定报告基因海肾萤光素酶的活性,分析不同突变对复制子复制水平的影响。结果表明,对C蛋白核定位序列进行突变均不同程度的提高了复制子报告基因的表达,提示登革病毒C蛋白核定位现象与病毒RNA复制有关,而此前并没有关于登革病毒C蛋白核定位功能的报道;缺失α1螺旋明显降低了复制子报告基因的表达,而缺失α2-α4螺旋对报告基因的表达水平没有明显的影响。进一步通过间接免疫荧光和实时定量RT-PCR在蛋白水平和核酸水平证实α1螺旋的缺失确实能够降低病毒RNA的复制效率,这表明C蛋白的α1螺旋或相应的编码序列在病毒RNA复制过程中发挥了一定作用。通过观察反式(trans)提供的C蛋白对缺失α1螺旋复制子复制的影响发现该复制子复制水平下降并非由于C蛋白结构的改变所致,而可能与C基因中的RNA二级结构的改变有关。该结果为进一步分析C基因及其编码蛋白在病毒RNA复制中的作用奠定了基础。
二、登革病毒C基因RNA二级结构在病毒RNA复制中的作用
为分析缺失α1螺旋的复制子复制水平下降是否由于C基因中的RNA二级结构元件被破坏所致,我们首先利用mfold软件对包括5′非编码区和C基因前109位核苷酸在内的登革病毒基因组5′端220nt的RNA序列进行了二级结构预测。结果表明,登革4型病毒C基因的54-98位核苷酸序列形成了一个在登革1-4型病毒中保守的二级结构(cα1HP),该二级结构由顶部茎环结构、左侧环、右侧环及基部双链区四个部分组成,并且该结构覆盖了α1螺旋编码区。为分析cα1HP对病毒RNA复制及蛋白翻译的影响,我们设计构建了一系列含有报告基因的突变体复制子,然后测定报告基因的表达水平,进而分析复制子复制和翻译水平的变化。结果表明,不同突变复制子在转染后6h的报告基因表达没有明显区别,说明cα1HP并不影响病毒的翻译过程。而对此二级结构的部分或全部序列进行缺失则均可导致病毒RNA复制水平的下降,说明病毒RNA的正常复制需要完整的cα1HP结构的存在。进一步通过引入沉默突变破坏碱基配对来改变cα1HP顶部茎环的二级结构使复制子的复制水平明显降低,而重新恢复碱基配对重建二级结构则使复制子的复制恢复到原型复制子的水平,对转染后不同时间点细胞中复制子RNA拷贝数进行实时定量RT-PCR分析,所得结果与报告基因表达活性测定结果是一致的。此外不改变RNA二级结构的突变体复制水平不发生改变。上述结果表明,cα1HP顶部的茎环结构对于病毒RNA的正常复制具有重要作用,并且其功能的发挥不依赖于核苷酸序列,而是依赖于二级结构本身的存在。
鉴于cα1HP中部左右两侧环的序列在登革1-4型病毒中高度保守,为分析cα1HP中部两侧环及基部双链区对病毒RNA复制的影响,我们又进一步分别针对左侧环、右侧环和基部双链区引入突变。结果发现,在左侧环中引入沉默突变使复制子的复制水平明显降低,而在右侧环和基部双链区引入突变对复制子的复制水平并没有影响,表明cα1HP左侧环保守序列的存在与维持病毒RNA的正常复制有关。
为了确认C蛋白α1螺旋本身与病毒RNA复制无关,我们在C基因的C73和C74之间插入一个U,同时删除原102位的C,目的是将C蛋白中α1螺旋的氨基酸序列完全移码突变,而保持RNA二级结构基本不变。结果显示,含有这一突变的复制子的复制水平与原型基本一致。这一结果进一步证实了C蛋白的α1螺旋本身并不影响病毒RNA复制的结论。
综上,我们在登革病毒复制子基础上对病毒C基因cα1HP的功能分析表明,cα1HP的存在能够促进病毒RNA的复制,且cα1HP保守的左侧环与其促进病毒复制的功能有关,其顶部的茎环结构在病毒RNA的正常复制中具有重要作用,并且这种作用的实现不依赖于核苷酸序列而是依赖于其二级结构本身。
三、与登革病毒C蛋白相互作用的病毒及宿主蛋白的筛选
在第一部分中,我们发现登革病毒C蛋白核定位与病毒RNA复制有关,这提示我们,登革病毒C蛋白可能具有包括调节病毒复制在内的多种不同的生物学功能。为了对登革病毒C蛋白影响病毒复制的分子基础进行探讨,我们利用CytoTrap酵母双杂交系统对与C蛋白可能存在相互作用的病毒非结构蛋白及宿主蛋白进行筛选。
本研究首先构建表达登革病毒C蛋白的诱饵载体以及分别表达登革病毒7种非结构蛋白(NS1,NS2A,NS2B,NS3,NS4A,NS4B和NS5)的重组穿梭质粒,然后分别共转化酵母cdc25H温度敏感株并观察其相互作用的情况。结果表明在酵母中病毒的C蛋白与其非结构蛋白间不存在直接的相互作用。为进一步筛选与登革病毒C蛋白可能存在相互作用的宿主蛋白,本研究分别以表达登革病毒C蛋白的酵母穿梭质粒及人脐带血内皮细胞cDNA文库作为诱饵载体和筛选文库。经筛选与反转实验,最终获得2株阳性克隆,经序列分析和同源性比对发现其中一株克隆的表达产物为人TARBP2蛋白。这为进一步探讨登革病毒C蛋白影响病毒复制的作用机制奠定了基础。
总之,本研究在登革病毒复制子基础上通过缺失突变发现C蛋白α1螺旋缺失可降低病毒RNA的复制效率,并通过RNA结构预测和突变分析证实C基因内的cα1HP结构对于维持病毒RNA的正常复制具有重要作用。同时本研究发现登革病毒C蛋白核定位现象与病毒RNA复制有关,并通过酵母双杂交筛选发现登革病毒C蛋白与人TARBP2蛋白存在相互作用。为深入阐明登革病毒C蛋白的功能和登革病毒的复制机制奠定了基础。
Dengue virus type 1-4, members of genus flavivirus, family flaviviridae, can cause dengue fever (DF) and more severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in humans. Dengue infections are becoming global public health concerns. The genome of dengue virus is a single-stranded RNA molecule of positive polarity comprising about 11,000 nucleotides, and it includes a long single open-reading frame, which is flanked by 5′and 3′untranslated region (UTR), encoding a polyprotein with about 3,300 ammo acids. Processing of the polyprotein results into 3 structural proteins(C, prM, and E) and at least 7 nonstructural proteins(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
Capsid (C) protein forms viral nucleocapsid via its interactions with viral genome RNA. C protein of flavivirus is composed of 4α-helixes, and it has been suggested to have multiple functions. C proteins of several members of the flaviviridae family act as modulators of viral RNA replication and protein translation, and the modulation effect of C protein on viral RNA replication is associated with its nuclear localization. Like C proteins of some other members of the flaviviridae family, Dengue virus C protein can be localized in the nuclei and nucleoli of infected cells, which implies it has potential function in viral RNA replication. Other than C protein itself, the gene of C protein also plays important roles in viral RNA replication and translation. C gene of flavivirus is adjacent to the 5′-UTR of the genomic RNA, which contains conserved RNA secondary structures essential for viral RNA replication. And RNA elements, 5′CS and cHP, which are critical for viral RNA replication, have been found in C gene. However, the mechanisms of how flavivirus C protein and RNA elements in C gene modulate viral replication are not understood very clearly yet, and in dengue virus, whether C protein also plays a role in viral replication is still unknown, and there is no answer to whether any other RNA secondary structure inside C gene modulates viral replication.
In the study, to investigate the effects of mutations in dengue virus C gene and its encoded protein on viral replication, a series of dengue virus replicon mutants were constructed and their replication efficiencies were compared. Furthermore, the role of RNA secondary structure in C gene in viral RNA replication was carefully studied in replicon system. Based on the correlation of dengue virus C protein’s nuclear localization and viral RNA replication, we performed yeast two-hybrid screen for viral and host proteins that interact with dengue virus C protein. Main results are described below.
1. Effects of C protein mutation on dengue virus RNA replication
In order to analyze whether any structures of dengue virus C protein have effects on viral replication, and the possibility of RNA secondary structure inside the coding sequence of C protein can modulate viral replication, we introduced various mutations into C gene of dengue virus replicon: the fourα-helix of C protein was deleted individually, and different mutations were introduced into the bipartite NLS of C protein. The replication of replicons was compared by measuring the reporter Renilla luciferase activities. Mutations that disabled the NLS of C protein increased viral RNA replication moderately. These results indicate that the nuclear localization of dengue virus C protein correlates with viral RNA replication, and this is the first report on the function of dengue virus C protein’s nuclear localization. Deletion ofα1-helix reduced viral RNA replication significantly, while deletion of the otherα-helixes did not affect viral RNA replication. Then by utilizing immunofluorescence assay and real-time quantitative RT-PCR, the effect ofα1-helix deletion on viral RNA replication was verified. Furthermore, we discovered the change of C protein structure did not account for the reduced replication ofΔα1 replicon by the co-transfection of a plasmid expressing C protein together withΔα1 replicon.
2. Role of RNA secondary structure in dengue virus C gene in viral RNA replication
To investigate whether the reduced replication ofΔα1 replicon was due to disruption of RNA secondary structures in C gene, first the structure of 5′220nt of dengue virus genome, including 5′-UTR and first 109nt of C gene, was predicted using mfold software. The 54-98nt region of DEN-4 C gene forms a long-stem-loop-like secondary structure which is conserved in all four dengue serotypes. This secondary structure, designated as cα1HP, covers the coding sequence of C proteinα1-helix and is composed of four sub-structures: the top hairpin, the left loop, the right loop and the bottom-paired region. A series of replicon mutants were constructed and reporter activities were measured to analyze the role of cα1HP in viral RNA replication and translation. The reporter activities of these mutants show no difference at 6h post transfection, which indicates disruption of cα1HP structure has no effect on viral translation. However, deletion of various parts of cHP, or the whole cα1HP structure reduces viral RNA replication, thus the integrity of cα1HP is required for normal viral RNA replication. Disruption of base pairs by introducing silent mutations into cα1HP top hairpin region reduces viral RNA replication, while restoration of cα1HP top hairpin recovers replication of the corresponding replicon. Analysis of replicon RNA copy numbers in transfected BHK-21 cells by quantitative RT-PCR consists with the results of reporter luciferase assay. Mutants such as MC69/70+82/83 and MC75/78, which don’t change cα1HP secondary structure, replicate at the same level as wild type replicon. Thus the top hairpin structure (65-87nt in C gene) of cα1HP is important for normal viral RNA replication and its function is sequence-independent.
The loop sequence in the middle of cα1HP is highly conserved in all four dengue serotypes. We introduced mutations into the left loop, the right loop and the bottom-paired region and observed the effect on viral RNA replication. Introducing silent mutations into left loop reduces viral RNA replication, while mutations in the right loop and the bottom-paired region have no effect on viral RNA replication. Thus the conserved sequence of left loop correlates with the maintaining of normal viral RNA replication.
We also constructed a frame-shift replicon mutant by inserting a U between C73 and C74 and deleting the former C102. In this mutant, theα1-helix of C protein is frame-shifted, whereas the overall RNA structure of the corresponding region is only slightly changed. This mutant shows similar replication effiency with wild type replicon. This finding confirms that theα1-helix itself has no effect on viral RNA replication.
Based on the afore mentioned results, we conclude that cα1HP can facilitate viral RNA replication and the conserved left loop correlates with its function in viral RNA replication, and the top hairpin of cα1HP also plays an important role in its regulation of viral RNA replication in a sequence-independent manner.
3. Screening for viral and host proteins that interact with dengue virus C protein
Previously, we have observed the nuclear localization of dengue virus C protein correlates with viral RNA replication. This implies C protein of dengue virus has multiple biological functions, including the modulation of viral replication. In order to investigate the role of C protein in viral replication, using CytoTrap yeast two-hybrid system, we performed a screen for viral and host proteins interacting with dengue virus C protein.
Dengue virus C gene was cloned into pSos vector, and dengue nonstructural genes were cloned into pMyr vector. The resulted recombinant plasmids were co-transformed into cdc25H, a temperature sensitive mutant of S.cerevisiae yeast and the potential interactions between dengue virus C protein and nonstructural proteins were checked. However, no direct interactions between these proteins were discovered. Host proteins that interact with dengue virus C protein were screened from human umbilical cord blood endothelial cells cDNA library. After screening and re-transforming, two positive clones were obtained, one of which was identified as human tarbp2 gene. These findings will contribute to further understandings of the mechanism of how dengue virus C protein modulates viral replication.
In summary, in this study we found deletion ofα1-helix of C protein reduces viral RNA replication using replicon system, and we have identified a new conserved secondary structure, the cα1HP, which plays an important role in viral RNA replication in C gene by RNA structure prediction and mutagenesis. And for the first time, we provided evidence that the nuclear localization of dengue virus C protein correlates with viral RNA replication, and dengue virus C protein interacts with TARBP2. This work contributes to the understanding of functions of dengue virus C protein and the replication mechanism of dengue virus.
黄病毒的衣壳(capsid,C)蛋白是病毒核衣壳的基本构成成分,多拷贝的C蛋白将病毒RNA包裹形成核衣壳结构。黄病毒C蛋白具有4个α螺旋结构(α1-α4),且该蛋白具有功能多样性。目前已经发现部分黄病毒的C蛋白对病毒RNA复制及蛋白翻译具有调节作用,并且C蛋白对病毒复制的调节与其核定位现象有关。登革病毒C蛋白的核定位现象提示我们,在登革病毒RNA复制过程中,C蛋白也可能发挥了一定功能。除C蛋白本身外,C基因在病毒RNA复制和翻译过程中也发挥了重要作用。黄病毒C基因紧邻与病毒复制密切相关的5′非编码区,而且在C基因中已经发现了多个在病毒复制和翻译过程中发挥重要作用的RNA结构元件。位于C基因内的5′环化序列(cyclization sequence, CS)和cHP元件对于病毒的复制和翻译均具有重要意义。但目前黄病毒C蛋白及C基因中的结构元件调节病毒复制的机制还不十分清楚。在登革病毒中,尚无C蛋白参与调节病毒复制的报道,而在C基因中是否存在其它影响病毒复制的RNA结构元件的问题也没有明确的答案。
针对上述问题,本研究利用病毒复制子技术分析登革病毒C基因及其编码蛋白突变对病毒RNA复制的影响,并通过与生物信息学手段相结合对C基因中的保守RNA二级结构在病毒RNA复制过程中的作用进行分析;在发现C蛋白核定位序列与病毒RNA复制有关的基础上,利用酵母双杂交技术筛选与C蛋白存在相互作用的病毒及宿主蛋白,从而对C蛋白参与调节病毒复制的作用机制进行探讨。研究内容主要包括以下三个部分。
一、登革病毒C蛋白基因突变对病毒RNA复制的影响
为分析登革病毒C蛋白中是否存在影响病毒复制的结构,并分析其基因中是否存在除5′CS和cHP外其它影响病毒复制的结构元件,我们构建了含有不同α螺旋编码序列缺失以及核定位序列突变的C基因的登革4型病毒复制子。然后通过测定报告基因海肾萤光素酶的活性,分析不同突变对复制子复制水平的影响。结果表明,对C蛋白核定位序列进行突变均不同程度的提高了复制子报告基因的表达,提示登革病毒C蛋白核定位现象与病毒RNA复制有关,而此前并没有关于登革病毒C蛋白核定位功能的报道;缺失α1螺旋明显降低了复制子报告基因的表达,而缺失α2-α4螺旋对报告基因的表达水平没有明显的影响。进一步通过间接免疫荧光和实时定量RT-PCR在蛋白水平和核酸水平证实α1螺旋的缺失确实能够降低病毒RNA的复制效率,这表明C蛋白的α1螺旋或相应的编码序列在病毒RNA复制过程中发挥了一定作用。通过观察反式(trans)提供的C蛋白对缺失α1螺旋复制子复制的影响发现该复制子复制水平下降并非由于C蛋白结构的改变所致,而可能与C基因中的RNA二级结构的改变有关。该结果为进一步分析C基因及其编码蛋白在病毒RNA复制中的作用奠定了基础。
二、登革病毒C基因RNA二级结构在病毒RNA复制中的作用
为分析缺失α1螺旋的复制子复制水平下降是否由于C基因中的RNA二级结构元件被破坏所致,我们首先利用mfold软件对包括5′非编码区和C基因前109位核苷酸在内的登革病毒基因组5′端220nt的RNA序列进行了二级结构预测。结果表明,登革4型病毒C基因的54-98位核苷酸序列形成了一个在登革1-4型病毒中保守的二级结构(cα1HP),该二级结构由顶部茎环结构、左侧环、右侧环及基部双链区四个部分组成,并且该结构覆盖了α1螺旋编码区。为分析cα1HP对病毒RNA复制及蛋白翻译的影响,我们设计构建了一系列含有报告基因的突变体复制子,然后测定报告基因的表达水平,进而分析复制子复制和翻译水平的变化。结果表明,不同突变复制子在转染后6h的报告基因表达没有明显区别,说明cα1HP并不影响病毒的翻译过程。而对此二级结构的部分或全部序列进行缺失则均可导致病毒RNA复制水平的下降,说明病毒RNA的正常复制需要完整的cα1HP结构的存在。进一步通过引入沉默突变破坏碱基配对来改变cα1HP顶部茎环的二级结构使复制子的复制水平明显降低,而重新恢复碱基配对重建二级结构则使复制子的复制恢复到原型复制子的水平,对转染后不同时间点细胞中复制子RNA拷贝数进行实时定量RT-PCR分析,所得结果与报告基因表达活性测定结果是一致的。此外不改变RNA二级结构的突变体复制水平不发生改变。上述结果表明,cα1HP顶部的茎环结构对于病毒RNA的正常复制具有重要作用,并且其功能的发挥不依赖于核苷酸序列,而是依赖于二级结构本身的存在。
鉴于cα1HP中部左右两侧环的序列在登革1-4型病毒中高度保守,为分析cα1HP中部两侧环及基部双链区对病毒RNA复制的影响,我们又进一步分别针对左侧环、右侧环和基部双链区引入突变。结果发现,在左侧环中引入沉默突变使复制子的复制水平明显降低,而在右侧环和基部双链区引入突变对复制子的复制水平并没有影响,表明cα1HP左侧环保守序列的存在与维持病毒RNA的正常复制有关。
为了确认C蛋白α1螺旋本身与病毒RNA复制无关,我们在C基因的C73和C74之间插入一个U,同时删除原102位的C,目的是将C蛋白中α1螺旋的氨基酸序列完全移码突变,而保持RNA二级结构基本不变。结果显示,含有这一突变的复制子的复制水平与原型基本一致。这一结果进一步证实了C蛋白的α1螺旋本身并不影响病毒RNA复制的结论。
综上,我们在登革病毒复制子基础上对病毒C基因cα1HP的功能分析表明,cα1HP的存在能够促进病毒RNA的复制,且cα1HP保守的左侧环与其促进病毒复制的功能有关,其顶部的茎环结构在病毒RNA的正常复制中具有重要作用,并且这种作用的实现不依赖于核苷酸序列而是依赖于其二级结构本身。
三、与登革病毒C蛋白相互作用的病毒及宿主蛋白的筛选
在第一部分中,我们发现登革病毒C蛋白核定位与病毒RNA复制有关,这提示我们,登革病毒C蛋白可能具有包括调节病毒复制在内的多种不同的生物学功能。为了对登革病毒C蛋白影响病毒复制的分子基础进行探讨,我们利用CytoTrap酵母双杂交系统对与C蛋白可能存在相互作用的病毒非结构蛋白及宿主蛋白进行筛选。
本研究首先构建表达登革病毒C蛋白的诱饵载体以及分别表达登革病毒7种非结构蛋白(NS1,NS2A,NS2B,NS3,NS4A,NS4B和NS5)的重组穿梭质粒,然后分别共转化酵母cdc25H温度敏感株并观察其相互作用的情况。结果表明在酵母中病毒的C蛋白与其非结构蛋白间不存在直接的相互作用。为进一步筛选与登革病毒C蛋白可能存在相互作用的宿主蛋白,本研究分别以表达登革病毒C蛋白的酵母穿梭质粒及人脐带血内皮细胞cDNA文库作为诱饵载体和筛选文库。经筛选与反转实验,最终获得2株阳性克隆,经序列分析和同源性比对发现其中一株克隆的表达产物为人TARBP2蛋白。这为进一步探讨登革病毒C蛋白影响病毒复制的作用机制奠定了基础。
总之,本研究在登革病毒复制子基础上通过缺失突变发现C蛋白α1螺旋缺失可降低病毒RNA的复制效率,并通过RNA结构预测和突变分析证实C基因内的cα1HP结构对于维持病毒RNA的正常复制具有重要作用。同时本研究发现登革病毒C蛋白核定位现象与病毒RNA复制有关,并通过酵母双杂交筛选发现登革病毒C蛋白与人TARBP2蛋白存在相互作用。为深入阐明登革病毒C蛋白的功能和登革病毒的复制机制奠定了基础。
Dengue virus type 1-4, members of genus flavivirus, family flaviviridae, can cause dengue fever (DF) and more severe dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in humans. Dengue infections are becoming global public health concerns. The genome of dengue virus is a single-stranded RNA molecule of positive polarity comprising about 11,000 nucleotides, and it includes a long single open-reading frame, which is flanked by 5′and 3′untranslated region (UTR), encoding a polyprotein with about 3,300 ammo acids. Processing of the polyprotein results into 3 structural proteins(C, prM, and E) and at least 7 nonstructural proteins(NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
Capsid (C) protein forms viral nucleocapsid via its interactions with viral genome RNA. C protein of flavivirus is composed of 4α-helixes, and it has been suggested to have multiple functions. C proteins of several members of the flaviviridae family act as modulators of viral RNA replication and protein translation, and the modulation effect of C protein on viral RNA replication is associated with its nuclear localization. Like C proteins of some other members of the flaviviridae family, Dengue virus C protein can be localized in the nuclei and nucleoli of infected cells, which implies it has potential function in viral RNA replication. Other than C protein itself, the gene of C protein also plays important roles in viral RNA replication and translation. C gene of flavivirus is adjacent to the 5′-UTR of the genomic RNA, which contains conserved RNA secondary structures essential for viral RNA replication. And RNA elements, 5′CS and cHP, which are critical for viral RNA replication, have been found in C gene. However, the mechanisms of how flavivirus C protein and RNA elements in C gene modulate viral replication are not understood very clearly yet, and in dengue virus, whether C protein also plays a role in viral replication is still unknown, and there is no answer to whether any other RNA secondary structure inside C gene modulates viral replication.
In the study, to investigate the effects of mutations in dengue virus C gene and its encoded protein on viral replication, a series of dengue virus replicon mutants were constructed and their replication efficiencies were compared. Furthermore, the role of RNA secondary structure in C gene in viral RNA replication was carefully studied in replicon system. Based on the correlation of dengue virus C protein’s nuclear localization and viral RNA replication, we performed yeast two-hybrid screen for viral and host proteins that interact with dengue virus C protein. Main results are described below.
1. Effects of C protein mutation on dengue virus RNA replication
In order to analyze whether any structures of dengue virus C protein have effects on viral replication, and the possibility of RNA secondary structure inside the coding sequence of C protein can modulate viral replication, we introduced various mutations into C gene of dengue virus replicon: the fourα-helix of C protein was deleted individually, and different mutations were introduced into the bipartite NLS of C protein. The replication of replicons was compared by measuring the reporter Renilla luciferase activities. Mutations that disabled the NLS of C protein increased viral RNA replication moderately. These results indicate that the nuclear localization of dengue virus C protein correlates with viral RNA replication, and this is the first report on the function of dengue virus C protein’s nuclear localization. Deletion ofα1-helix reduced viral RNA replication significantly, while deletion of the otherα-helixes did not affect viral RNA replication. Then by utilizing immunofluorescence assay and real-time quantitative RT-PCR, the effect ofα1-helix deletion on viral RNA replication was verified. Furthermore, we discovered the change of C protein structure did not account for the reduced replication ofΔα1 replicon by the co-transfection of a plasmid expressing C protein together withΔα1 replicon.
2. Role of RNA secondary structure in dengue virus C gene in viral RNA replication
To investigate whether the reduced replication ofΔα1 replicon was due to disruption of RNA secondary structures in C gene, first the structure of 5′220nt of dengue virus genome, including 5′-UTR and first 109nt of C gene, was predicted using mfold software. The 54-98nt region of DEN-4 C gene forms a long-stem-loop-like secondary structure which is conserved in all four dengue serotypes. This secondary structure, designated as cα1HP, covers the coding sequence of C proteinα1-helix and is composed of four sub-structures: the top hairpin, the left loop, the right loop and the bottom-paired region. A series of replicon mutants were constructed and reporter activities were measured to analyze the role of cα1HP in viral RNA replication and translation. The reporter activities of these mutants show no difference at 6h post transfection, which indicates disruption of cα1HP structure has no effect on viral translation. However, deletion of various parts of cHP, or the whole cα1HP structure reduces viral RNA replication, thus the integrity of cα1HP is required for normal viral RNA replication. Disruption of base pairs by introducing silent mutations into cα1HP top hairpin region reduces viral RNA replication, while restoration of cα1HP top hairpin recovers replication of the corresponding replicon. Analysis of replicon RNA copy numbers in transfected BHK-21 cells by quantitative RT-PCR consists with the results of reporter luciferase assay. Mutants such as MC69/70+82/83 and MC75/78, which don’t change cα1HP secondary structure, replicate at the same level as wild type replicon. Thus the top hairpin structure (65-87nt in C gene) of cα1HP is important for normal viral RNA replication and its function is sequence-independent.
The loop sequence in the middle of cα1HP is highly conserved in all four dengue serotypes. We introduced mutations into the left loop, the right loop and the bottom-paired region and observed the effect on viral RNA replication. Introducing silent mutations into left loop reduces viral RNA replication, while mutations in the right loop and the bottom-paired region have no effect on viral RNA replication. Thus the conserved sequence of left loop correlates with the maintaining of normal viral RNA replication.
We also constructed a frame-shift replicon mutant by inserting a U between C73 and C74 and deleting the former C102. In this mutant, theα1-helix of C protein is frame-shifted, whereas the overall RNA structure of the corresponding region is only slightly changed. This mutant shows similar replication effiency with wild type replicon. This finding confirms that theα1-helix itself has no effect on viral RNA replication.
Based on the afore mentioned results, we conclude that cα1HP can facilitate viral RNA replication and the conserved left loop correlates with its function in viral RNA replication, and the top hairpin of cα1HP also plays an important role in its regulation of viral RNA replication in a sequence-independent manner.
3. Screening for viral and host proteins that interact with dengue virus C protein
Previously, we have observed the nuclear localization of dengue virus C protein correlates with viral RNA replication. This implies C protein of dengue virus has multiple biological functions, including the modulation of viral replication. In order to investigate the role of C protein in viral replication, using CytoTrap yeast two-hybrid system, we performed a screen for viral and host proteins interacting with dengue virus C protein.
Dengue virus C gene was cloned into pSos vector, and dengue nonstructural genes were cloned into pMyr vector. The resulted recombinant plasmids were co-transformed into cdc25H, a temperature sensitive mutant of S.cerevisiae yeast and the potential interactions between dengue virus C protein and nonstructural proteins were checked. However, no direct interactions between these proteins were discovered. Host proteins that interact with dengue virus C protein were screened from human umbilical cord blood endothelial cells cDNA library. After screening and re-transforming, two positive clones were obtained, one of which was identified as human tarbp2 gene. These findings will contribute to further understandings of the mechanism of how dengue virus C protein modulates viral replication.
In summary, in this study we found deletion ofα1-helix of C protein reduces viral RNA replication using replicon system, and we have identified a new conserved secondary structure, the cα1HP, which plays an important role in viral RNA replication in C gene by RNA structure prediction and mutagenesis. And for the first time, we provided evidence that the nuclear localization of dengue virus C protein correlates with viral RNA replication, and dengue virus C protein interacts with TARBP2. This work contributes to the understanding of functions of dengue virus C protein and the replication mechanism of dengue virus.
引文
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[3] Valle RP, Falgout B. Mutagenesis of the NS3 protease of dengue virus type 2. J Virol, 1998, 72(1):624-632.
[4] Matusan AE, Kelley PG, Pryor MJ, et al. Mutagenesis of the dengue virus type 2 NS3 proteinase and the production of growth-restricted virus. J Gen Virol, 2001, 82(Pt 7):1647-1656.
[5] Amberg SM, Rice CM. Mutagenesis of the NS2B-NS3-mediated cleavage site in the flavivirus capsid protein demonstrates a requirement for coordinated processing. J Virol, 1999, 73:8083–8094.
[6] Lobigs M, Lee E. Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes. J Virol, 2004, 78(1):178-186.
[7] Markoff L, Falgout B, Chang A. A conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein. Virology, 1997, 233(1):105-117.
[8] Ma L, Jones CT, Groesch TD, et al. Solution structure of dengue virus capsid protein reveals another fold. PNAS, 2004, 101(10):3414-3419.
[9] Patkar CG, Jones CT, Chang YH, et al. Functional requirements of the yellow fever virus capsid protein. J Virol, 2007, 1(12):6471-6481.
[10] Mori Y, Okabayashi T, Yamashita T, et al. Nuclear localization of Japanese encephalitis virus core protein enhances viral replication. J Virol, 2005, 79(6):3448-3458.
[11] Tsuda Y, Mori Y, Abe T, et al. Nucleolar protein B23 interacts with Japanese encephalitis virus core protein and participates in viral replication. Microbiol Immunol, 2006, 50(3):225-234.
[12] Mori Y, Yamashita T, Tanaka Y, et al. Processing of Capsid Protein by Cathepsin L Plays a Crucial Role in Replication of the Japanese Encephalitis Virus in Neural and Macrophage Cells. J Virol, 2007, 81(16):8477-8487.
[13] Westaway EG, Khromykh AA, Kenney MT, et al. Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus. Virology, 1997, 234(1):31-41.
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