RNA病毒翻译调控元件—内部核糖体进入位点(IRES)

真核生物大多数蛋白质合成采用了依赖帽子结构的翻译起始方式.但一组缺乏帽子构的RNA病毒的蛋白质合成起始是依赖其5′端非翻译区（untranslated region,UTR）翻译调控的顺式作用元件——内部核糖体进入位点（internal ribosome entry site, IRES）.它 们能够在一些反式作用因子的辅助下,招募核糖体小亚基到病毒mRNA的翻译起始位点.前,依赖IRES元件翻译起始的RNA病毒在哺乳动物,无脊椎动物及植物中均有发现.因此,对RNA病毒IRES元件的深入研究,不仅有助于阐明相关疾病的发生机理,而且为工业应用和疾病治疗提供借鉴意义.本文对RNA病毒IRES元件发现、分类、结构与功能等作了综述.     

内部核糖体进入位点研究现状及应用展望

内部核糖体进入位点 (ｉｎｔｅｒｎａｌｒｉｂｏｓｏｍｅｅｎｔｒｙｓｉｔｅ ,ＩＲＥＳ)是最早发现于动物病毒基因组 5′非编码区的一段ＤＮＡ序列 ,它具有不依赖于 5′帽子结构的翻译起始功能。1 .ＩＲＥＳ的发现基因的表达分为转录和翻译两个相互独立但又紧密联系的阶段。正常情况下真核细胞的ｍＲＮＡ前体转录完成后 ,经过剪接、5′端加帽、3′端加尾等修饰过程生成成熟的ｍＲＮＡ。 5′帽子结构除了能使ｍＲＮＡ免遭核酸酶和磷酸酶的攻击 ,在随后的翻译起始中也起到十分重要的作用。核糖体小亚基通过识别ｍＲＮＡ 5′端帽子结构来寻找蛋…     

内部核糖体进入位点及其研究进展

内部核糖体进入位点(IRES)是mRNA5'端非编码区的一段特殊序列,允许核糖体直接在此序列结合mRNA并起始翻译。对IRES的发现、分类、特征,以及细胞中是否存在该元件进行了简要综述。     

丙肝病毒内部核糖体进入位点Ⅲd区域及其适配体的发夹结构

发夹结构于RNA的生物学功能有重要影响.该文从实验和理论预测结构两方面,介绍丙肝病毒（HCV）内部核糖体进入位点（IRES）Ⅲd区域及其适配体（aptamer）RNA中的发夹结构在翻译起始时的作用.     

口蹄疫病毒基因组内部核糖体进入位点一级和二级结构分析

以口蹄疫病毒(foot-and-mouth disease virus,FMDV)强毒China/99株牛舌水泡皮为材料,用RT-PCR法提取RNA及扩增目的cDNA,然后与pGEM-T Easy载体连接并转化JM109菌株,再经重组质粒电泳、PCR和EcoRI酶切鉴定.用DNAstar软件比较了内部核糖体进入位点(IRES)的序列差异,并用RNAdraw软件绘制和分析了该区段的二级结构.8株FMDV IRES核苷酸序列比较表明该区段较为保守,并对非保守区域进行了分析.二级结构分析表明,FMDV IRES至少有3种二级结构图形:第一型有5个结构域,与Pilipenko等报道的一致;第二和三型分别有6和11个结构域,与Pilipenko等报道的结果不同.无论FMDV IRES二级结构如何不同,但单链区大部分核苷酸序列或基序相同,如AACUCC、GAAA、CUUU、AGG、AACC、GUAA等.茎环柄部核苷酸对维持二级结构的空间构像具有十分重要的作用,环中或单链区序列(基序)在维持其功能方面具有很重要的作用,如GAAA和CUUU基序分别是三级结构的组件和嘧啶区结合蛋白的结合位点.     

内部核糖体进入位点（IRES）调控细胞血清饥饿应激条件下PRDM13的翻译

PRDM13是锌指蛋白转录抑制因子（positive regulatory domain zinc finger protein,PRDM) 家族中的一员,其在细胞分化、肿瘤的发生和恶性转化中起着重要的作用。而对于PRDM13 基因侧翼序列是否含有内部核糖体进入位点(internal ribosome entry site, IRES）及其功能所知甚少。本研究对PRDM13 5′端的非翻译区（5′UTR）进行IRES结构与功能分析,探索其在细胞血清饥饿应激条件下对PRDM13翻译的影响。实验发现,在血清饥饿的条件下,肝癌细胞Bel7402/WT中PRDM13的蛋白质水平增加,但是其mRNA水平基本没有变化。将PRDM13 5′UTR的序列插入双顺反子的报告载体(pRL-FL)中,并且将构建的载体(pRL-PRDM13-FL）转染进细胞中,结果显示,PRDM13 5′UTR含有IRES,且发现PRDM13 5′UTR中的105 nt（53～157）对其IRES的功能至关重要。在帽依赖的翻译（cap-dependent translation）机制被抑制时,IRES这种机制可有效维持PRDM13蛋白合成。本研究提供了在细胞压力条件下调节PRDM13蛋白合成的一种新的解释。     

应用绿色荧光蛋白研究外源基因在造血细胞的表达调控

以增强型绿色荧光蛋白为报道分子,研究双基因逆转录病毒载体介导小鼠骨髓细胞的基因转移中,SV(simianvirus)40启动子和内部核糖体进入位点(internalribosomeentrysite,IRES)对双基因共表达的影响。构建双基因中间序列分别为SV40启动子和IRES的载体pLESN和pLEIN。经包装获得较高滴度的病毒上清,以共培养的方式感染5-氟尿嘧啶预刺激的小鼠骨髓细胞。流式细胞仪检测表明转染效率约25%,PCR证明EGFP基因整合至骨髓细胞基因组。半固体培养转基因细胞7d,LEIN组获得具有G418抗性的集落中98%表达绿色荧光,而LESN组54%。结果表明:在双基因逆转录病毒载体介导的小鼠骨髓细胞的基因转导中,IRES与内部SV40启动子相比,更能保证双基因的共同表达。     

转录激活因子1(ATF1)内部核糖体进入位点的鉴定及活性分析

蛋白质翻译起始通常有两种机制,一是依赖帽结构的翻译,另一种是依赖5′非翻译区的内部核糖体进入位点(IRES).在后一种方式中,在某些IRES反式作用因子,如La蛋白、多聚嘧啶串结合蛋白1等的参与下,直接招募核糖体小亚基到mRNA的翻译起始位点,启始翻译.研究发现,参与细胞生长、分化、细胞周期进程、凋亡和压力调控的相关蛋白中通常含有IRES元件.基于功能,我们提出假说：转录激活因子1(ATF1)的5′-UTR可能具有IRES活性.为验证假说,首先构建了含全长ATF1 5′-UTR的双荧光素酶报告质粒|质粒转染结合报告酶活性分析显示,ATF1 5′-UTR在Bel7402、HCT-8和HEK293细胞中表现出不同的IRES活性|而此IRES活性与5′-UTR中的隐藏启动子无关.同时还发现,ATF1 5′-UTR在NIH3T3细胞中却没有IRES活性.与此结果相一致,Western印迹检测ATF1在这几种细胞系中的表达.结果显示,Bel7402、HCT 8和HEK293中ATF1蛋白质表达水平较高,而在NIH3T3中却极低. ATF1 5′-UTR的系列5′-删除突变及报告酶分析证明,ATF1 5′-UTR的完整性对其IRES活性大小发挥重要作用|其中5′端的204 bp序列对其IRES活性贡献较大. RNA-蛋白免疫共沉淀实验揭示,ATF1 5′-UTR可与La和PTBP1蛋白结合|抑制La和PTBP1蛋白质的表达,并可减低HEK293细胞中ATF1蛋白质表达水平.这些结果提示,La和PTBP1蛋白（两种ITAFs）为ATF1 5′-UTR发挥IRES活性所必需.总之,上述结果证明,ATF1 5′-UTR具有IRES活性,其活性发挥依赖与La和PTBP1蛋白的结合.上述发现为进一步研究La和PTBP1表达及亚细胞定位对ATF1 IRES调控机制的影响奠定了基础.     

用于筛选抑制丙型肝炎病毒内部核糖体进入位点活性药物的整合型细胞药物筛选模型的构建和评价

目的：构建一个能用于筛选抑制丙型肝炎病毒内部核糖体进入位点（HCV—IRES）活性药物的整合型细胞药筛模型。方法：构建携带HCV 5＇-UTR调控报告基因分泌型碱性磷酸酶（SEAP）基因的质粒pHCV5’-SEAP和用作筛选标记的pNeo^R,线性化后共转染于Huh-7细胞中,经G418筛选后得到抗性细胞克隆,进行SEAP定量检测筛选后,得到能用于筛选抑制HCV-IRES活性药物的整合型细胞药筛模型;连续培养24代,用MTT法测细胞相对活力评价该药筛模型的生长稳定性,用SEAP定量检测法评价该药筛模型的SEAP表达稳定性,用反义寡聚核苷酸抑制试验评价模型的药筛灵敏度。结果：G418筛选后得到26个抗性细胞克隆;对其中5个抗性细胞克隆进行SEAP定量检测筛选后,得到3个能表达SEAP的阳性细胞克隆;连续培养24代过程中,该药筛模型的生长稳定性大干80％,SEAP表达稳定性达95％。结论：构建的细胞模型具有良好的稳定性和药筛灵敏度,符合作为药物筛选模型的要求。     

肠道病毒71型(EV71)荧光病毒的构建

人肠道病毒71型(Enterovirus,EV71)是引起手足口病的主要病原体。首先利用反向遗传学的方法,构建了EV71全长cDNA感染性克隆,经体外转录、转染RD细胞后成功获得了拯救病毒。随后,将增强型绿色荧光蛋白基因(Enhanced green fluorescent protein,EGFP)插入到EV71基因组中构建荧光病毒。结果表明,此荧光病毒不仅可以侵染、复制,在传代的过程中EGFP基因也保持了较好的稳定性,表明其可以作为一种报告病毒应用于高通量的EV71抗病毒药物筛选中。     

Effects of magnesium ions on two EMCV IRES key activity fragments

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino protons, changes have been monitored in base-pair kinetics induced by Mg^2 + in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus internal ribosome entry site. For r(CACCUGGCGACAGGUG), the addition of Mg^2 + reveals two types of base-pairs: r(U⁵⁴⁵·A) and r(G⁵⁴⁶·C), in the first category, have lifetimes only slightly higher in the presence of Mg^2 +, whereas their dissociation constants are substantially reduced. This behavior has been termed proximal. The base-pairs r(G⁵⁵³·C) and r(G⁵⁵⁴·C), in the second category, have lifetimes substantially higher in the presence of Mg^2 +, whereas their dissociation constants remain almost constant. This behavior has been termed distal. Mg^2 + has a specific effect on r(CACCUGGCGACAGGUG), the magnitude of which is progressively modulated from the proximal region of the 16-mer towards its distal region. For r(GGCCAAAAGCC), an intermediate behavior is found for base-pairs r(G⁵⁶⁵·C) and r(G⁵⁷²·C). Their lifetimes are slightly higher in the presence of Mg^2 + and their dissociation constants are significantly lower, a behavior resembling that of the 16-mer proximal region. These results indicate that Mg^2 + diffusively moves around r(GGCCAAAAGCC).     

小RNA病毒蛋白翻译调控元件研究进展

真核生物的起始复合物并不是在起始AUG处形成 ,而是在mRNA的 5′末端形成 ,其识别信号就是 5′末端的帽子结构。小RNA病毒科成员RNA 5′末端没有帽子结构 ,而有一个病毒编码的小蛋白质与基因组共价相连。小RNA病毒的蛋白翻译起始于 5′非翻译区中的内部顺式调控元件 ,称为内部核糖体进入位点 (IRES)。口蹄疫病毒 (foot and mouthdiseasevirus,FMDV)是该科病毒的典型代表 ,引起偶蹄动物的急性接触性传染病。完整FMDV含有单链正股RNA、衣壳蛋白及少量装配过程中夹带的非结构蛋白和宿主细胞肌动蛋白 ,其基因组RNA全长约 8 5kb ,可直接作为信使RNA。对IRES的一、二级结构进行了比较 ,对IRES与翻译起始因子的相互作用以及对病毒毒力的影响作了综述。     

Comparison of impact of EMCV replication on the nuclear apparatus of NIH 3T3 and HEp-2 cells

We have investigated differences between the actions of encephalomyocarditis virus (EMCV) on cytometric indices in cultured NIH 3T3 and HEp-2 cells, which are characterized by different levels of transformation. HEp-2 cells surviving 48 h after EMCV infection showed lower nuclear ploidy, reduced nuclear area, fewer nucleoli and a higher percentage of euploid cells. There was a significant increase of nucleolar/nuclear DNA 6-24 h after EMCV infection. However, EMCV had markedly different effects on NIH 3T3 cells: there was a consistent increase in population ploidy, but the average number of nucleoli and the number of euploid cells in the population remained constant. The nucleolar/nuclear DNA ratio was almost unchanged. These different viral effects might be explained by the contrasting levels of differentiation of the cultured cell lines. The number of nucleoli does not depend on the amount of nuclear DNA in either viral-infected or intact cells but on the euploidy-to-aneuploidy ratio. The ratio of the sums of the nucleolar perimeters to the nuclear perimeter increases linearly with the number of nucleoli per nucleus in both intact and virus-infected cells. In both cell lines, the amount of DNA per nucleolus decreases as the number of nucleoli increases.     

Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location

The internal ribosomal entry site (IRES)from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Compared head-to-head with dual-luciferase reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a similarly configured pIRES vector. It also produced nearly twice as much protein as pCITE-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. The preferred IRES (viral bases 273-845) and the minimum IRES (viral bases 400-836) for optimum activity are illustrated.     

Identification of the internal ribosome entry sites (IRES) of prion protein gene

Many studies demonstrated that there are several type bands of prion protein in cells. However, the formation of different prion protein bands is elusive. After several low molecular weight bands of prion protein appeared in SMB-S15 cells infected with scrapie agent Chandler, we think that IRES-dependent translation mechanism induced by prion is involved in the formation of prion protein bands. Then we designed a series of pPrP-GFP fusing plasmids and bicistronic plasmids to identify the IRES sites of prion protein gene and found 3 IRES sites inside of PrP mRNA. We also demonstrated that cap-independent translation of PrP was associated with the ER stress through Tunicamycin treatment. We still found that only IRE1 and PERK pathway regulated the IRES-dependent translation of PrP in this study. Our results indicated, we found that PrP gene had an IRES-dependent translation initiation mechanism and we successfully identified the IRESs inside of the prion protein gene.     

Internal translation initiation on the foot-and-mouth disease virus IRES is affected by ribosomal stalk conformation

A human cell line, in which expression of the ribosomal stalk proteins P1 and P2 has been suppressed by RNAi technology, has been used to test how the loss of these proteins affects IRES-dependent translation. Foot-and-mouth disease virus (FMDV) IRES-dependent translation from a bicistronic construct is about three fold higher in the P1/P2-depleted cells than in control cells in the presence of Lb protease. By contrast, no effect on Hepatitis C virus (HCV) IRES translation was observed. These results emphasize the functional heterogeneity of the IRES and they highlight a functional connection between the ribosomal stalk and picornavirus IRES-dependent translation.     

核酸疫苗双表达载体的构建及表达

将微小病毒内部核糖体进入位点(IRES)基因克隆到质粒pVAXI载体多克隆位点,构建出核酸疫苗双表达载体pVI。将绿色荧光蛋白(EGFP)基因和新霉素磷酸转移酶(neor)基因作为报告基因,连接到pVI载体IRES基因的前后两处多克隆位点,构建出表达载体pEIN。通过脂质体介导的方法将该载体转染COS-7细胞,筛选到同时表达绿色荧光蛋白和新霉素磷酸转移酶的表达株,表明成功地构建了核酸疫苗双表达载体,为构建多价核酸疫苗及带有分子佐剂的核酸疫苗打下了基础。