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。     

Caveolin-1在EMCV感染宿主细胞中的作用

脑心肌炎病毒(EMCV)是一种重要的人畜共患病病原,但其致病机制目前尚不明确,小窝蛋白-1(Caveolin-1)可介导多种病毒感染宿主细胞。为了探究Caveolin-1在EMCV感染宿主细胞中的作用,该实验检测了EMCV感染不同时间段He La细胞中Caveolin-1的表达量,对He La细胞和HEK-293细胞中Caveolin-1进行瞬时过表达实验、瞬时沉默实验和Caveolin-1依赖型内吞途径抑制实验,然后观察EMCV对He La细胞和HEK-293细胞的感染情况。结果发现, HeLa细胞中Caveolin-1表达量会随病毒感染时间的增加而升高;在Caveolin-1瞬时过表达实验结果中,病毒滴度、病毒拷贝数、病毒蛋白检测结果均显示,上调Caveolin-1促进EMCV感染宿主细胞, Caveolin-1瞬时沉默实验和Caveolin-1依赖型内吞途径抑制实验的结果则显示,下调Caveolin-1或抑制Caveolin-1依赖型内吞途径可抑制EMCV感染宿主细胞。上述现象提示, EMCV可通过Caveolin-1依赖型内吞途径感染宿主细胞。     

口蹄疫病毒与宿主细胞的相互作用研究进展

口蹄疫病毒（FMDV）导致了偶蹄动物口蹄疫的发生,它是一类有着自身特点的RNA病毒。首先,FMDV衣壳蛋白VP1识别结合宿主细胞膜上的整联蛋白等受体,以内吞的方式进入细胞,利用宿主细胞成分完成病毒蛋白的合成。这些新合成的L^pro、2C和3C^pro等病毒致病因子进一步抑制宿主基因的转录和翻译,诱导细胞凋亡和白噬,并抑制干扰素介导的一系列先天性和获得性免疫反应。宿主则在病毒侵染细胞的初期,利用病毒识别受体等来识别病毒并诱导合成干扰素等细胞因子,介导多种免疫反应以清除病毒。病毒和宿主两者在持续的利用和较量中完成疾病的发生和痊愈等。其次,不断发现的病毒受体、结合基序、致病因子及宿主细胞的多种免疫调节因子将成为相关领域新的研究内容。综上,开发高效安全疫苗、增强自身免疫力及利用RNAi直接抑制病毒RNA等便成为现代FMDV防治的主要内容。     

丙型肝炎病毒5

丙型肝炎病毒(HCV)RNA5′-非编码区(5′-NTR)由341个核苷酸组成,形成4个茎-环二级结构,5′-NTR二级结构及某些部分单链序列的核苷酸组成是病毒翻译起始的先决条件.5′-NTR中的大部分核苷酸序列组成内部核糖体进入位点(IRES),在宿主细胞蛋白质因子La自身抗原、eIF3、多聚嘧啶区结构蛋白(PTB)、多聚胞嘧啶结合蛋白(PCBP-1、2)等的作用下,形成复杂的翻译起始复合物,对HCV的翻译过程进行精确调控,完成帽状结构形成非依赖性的蛋白翻译过程.HCV RNA 5′-NTR翻译过程的分子生物学机制的研究,将有助于HCV治疗新方法和新途径的探索.     

丙型肝炎病毒5′-非编码区及其结合蛋白的研究进展

丙型肝炎病毒(HCV)RNA5′-非编码区(5′-NTR)由341个核苷酸组成,形成4个茎-环二级结构,5′-NTR二级结构及某些部分单链序列的核苷酸组成是病毒翻译起始的先决条件.5′-NTR中的大部分核苷酸序列组成内部核糖体进入位点(IRES),在宿主细胞蛋白质因子La自身抗原、eIF3、多聚嘧啶区结构蛋白(PTB)、多聚胞嘧啶结合蛋白(PCBP-1、2)等的作用下,形成复杂的翻译起始复合物,对HCV的翻译过程进行精确调控,完成帽状结构形成非依赖性的蛋白翻译过程.HCV RNA 5′-NTR翻译过程的分子生物学机制的研究,将有助于HCV治疗新方法和新途径的探索.     

HSP27过表达抑制脑心肌炎病毒复制的作用初探

已有研究表明,HSP27在一些病毒的生命周期中发挥着重要作用,但它对于脑心肌炎病毒(encephalomyocarditis virus,EMCV)的调控作用尚不明晰。该研究通过构建人源HSP27的表达质粒pCMV-Myc-HSP27并于HEK293细胞中表达后,接种EMCV检测病毒的复制及相关通路蛋白表达情况。结果显示,过表达HSP27可以抑制EMCV在宿主细胞中的复制,进一步分析表明,HSP27可能是通过正调控IFN-β信号通路中接头分子MAVS、TBK1、IRF3的表达和阻止自噬体与溶酶体的融合实现对EMCV复制的负调控作用。总之,该研究首次表明,HSP27抑制EMCV复制是通过IFN-β信号通路及自噬途径来实现的,这些发现为揭示EMCV感染中宿主因子的调控作用和潜在的抗病毒靶点提供新的见解。     

膜联蛋白A2对脑心肌炎病毒在BHK-21细胞中增殖的影响

膜联蛋白A2是一种参与调节多种病毒增殖的重要宿主蛋白。本研究通过构建过表达膜联蛋白A2的BHKAnxa2细胞系及瞬时敲低膜联蛋白A2,探讨膜联蛋白A2对脑心肌炎病毒在BHK-21细胞中增殖的影响。通过RT-PCR从BHK-21细胞中扩增膜联蛋白A2全长cDNA,测序正确后克隆入pcDNA3.1整合表达载体中;用重组载体pcDNA3.1-Anxa2转染BHK-21细胞,经G418筛选获得抗性克隆,qRT-PCR和Western blot试验证明BHKAnxa2过表达膜联蛋白A2;EMCV感染试验证明BHK-Anxa2细胞中病毒滴度高于对照组。通过siRNA降低BHK-21细胞中膜联蛋白A2表达,EMCV感染试验证明膜联蛋白A2表达下降后EMCV增殖也随之下降。以上试验结果表明鼠膜联蛋白A2表达改变可导致病毒在BHK-21细胞中的增殖发生变化,提示膜联蛋白A2与EMCV在BHK-21细胞中的增殖相关。     

病毒受体的研究方法

1概况 病毒受体可以定义为位于宿主细胞表面能够被病毒吸附蛋白识别并与之结合,从而引起病毒感染的分子复合物,其化学本质是糖蛋白、蛋白聚糖、脂类或糖脂,大多数属于蛋白质.病毒受体可以是单体也可以是多分子复合物,具有特异性、高亲和性、饱和性、结合位点及靶细胞部位的有限性以及独特的生物学活性等[1].病毒受体是公认的引发病毒感染宿主细胞的主要决定因素,也是影响病毒宿主特异性和组织亲嗜性的决定因素之一.     

动物宿主microRNAs抗病毒作用机制研究进展

microRNAs(miRNAs)是一种长度为22nt的非编码RNA分子,能够结合mRNA,影响翻译效率,调控蛋白的表达。近来多项研究表明,动物宿主miRNAs通过多种机制抑制病毒的复制,如直接靶向病毒基因、干扰病毒复制所需因子、调节先天性免疫、调节细胞凋亡、调节细胞免疫,发挥抗病毒效应。本文归纳总结宿主miRNAs对病毒复制的调控机制,讨论miRNAs类抗病毒药物的应用情况,并展望该类药物的发展趋势。     

Direct evidence that polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA.

The requirement of PTB, polypyrimidine tract binding protein, for internal initiation of translation has been tested using an RNA affinity column to deplete rabbit reticulocyte lysates of PTB. The affinity column was prepared by coupling CNBr-activated Sepharose with the segment of the 5'-untranslated region of encephalomyocarditis virus (EMCV) RNA previously shown to bind PTB. Lysates passed through this column were devoid of PTB, and were incapable of internal initiation of translation dependent on the EMCV 5'-untranslated region, while retaining the capacity for translation dependent on ribosome scanning. Full activity for internal initiation was restored by the addition of recombinant PTB at the physiologically relevant concentration of about 5 micrograms/mL. When various PTB deletion mutants were tested, it was found that this activity required virtually the full-length protein. Thus, PTB is an essential protein for internal initiation promoted by the EMCV 5'-untranslated region. However, the PTB-depleted lysate retained the capacity for internal initiation promoted by the 5'-untranslated regions of another cardiovirus, Theiler's murine encephalomyelitis virus, and of the unrelated hepatitis C virus, and in neither case did addition of recombinant PTB stimulate internal initiation. Therefore, PTB is not a universal internal initiation factor that is indispensable in every case of internal ribosome entry.     

Conserved nucleotides within the J domain of the encephalomyocarditis virus internal ribosome entry site are required for activity and for interaction with eIF4G

The internal ribosome entry site (IRES) elements of cardioviruses (e.g., encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus) are predicted to have very similar secondary structures. Among these complex RNA structures there is only rather limited complete sequence conservation. Within the J domain of the EMCV IRES there are four highly conserved nucleotides (A704, C705, G723, and A724)., which are predicted to be unpaired and have been targeted for mutagenesis. Using an IRES-dependent cell selection system, we have isolated functional IRES elements from a pool of up to 256 mutants. All changes to these conserved nucleotides resulted in IRES elements that were less efficient at directing internal initiation of translation than the wild-type element, and even some of the single point mutants were highly defective. Each of the mutations adversely affected the ability of the RNAs to interact with the translation initiation factor eIF4G.     

NMD resulting from encephalomyocarditis virus IRES-directed translation initiation seems to be restricted to CBP80/20-bound mRNA

Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely.     

Initiation of protein synthesis by internal entry of ribosomes into the 5' nontranslated region of encephalomyocarditis virus RNA in vivo.

Expression vectors that yield mono-, di-, and tricistronic mRNAs upon transfection of COS-1 cells were used to assess the influence of the 5' nontranslated regions (5'NTRs) on translation of reporter genes. A segment of the 5'NTR of encephalomyocarditis virus (EMCV) allowed translation of an adjacent downstream reporter gene (CAT) regardless of its position in the mRNAs. A deletion in the EMCV 5'NTR abolishes this effect. Poliovirus infection completely inhibits translation of the first cistron of a dicistronic mRNA that is preceded by the capped globin 5'NTR, whereas the second cistron preceded by the EMCV 5'NTR is still translated. We conclude that the EMCV 5'NTR contains an internal ribosomal entry site that allows cap-independent initiation of translation. mRNA containing the adenovirus tripartite leader is also resistant to inhibition of translation by poliovirus.     

The polypyrimidine tract binding protein (PTB) requirement for internal initiation of translation of cardiovirus RNAs is conditional rather than absolute.

Picornavirus RNAs are translated by an unusual mechanism of internal ribosome entry that requires a substantial segment of the viral 5'-untranslated region, generally known as the internal ribosome entry segment (IRES), and in some circumstances may require cellular trans-acting proteins, particularly polypyrimidine tract binding protein (PTB). It is shown here that for encephalomyocarditis virus (EMCV), the PTB dependence of IRES function in vitro is determined partly by the nature of the reporter cistron, and more especially by the size of an A-rich bulge in the IRES. With a wild-type EMCV IRES (which has a bulge of 6 As), translation is effectively independent of PTB provided the IRES is driving the synthesis of EMCV viral polyprotein. With an enlarged (7A) bulge and heterologous reporters, translation is highly dependent on PTB. Intermediate levels of PTB dependence are seen with a 7A bulge IRES driving viral polyprotein synthesis or a wild-type (6A) bulge IRES linked to a heterologous reporter. None of these parameters influenced the binding of PTB to the high-affinity site in the IRES. These results argue that PTB is not an essential and universal internal initiation factor, but, rather, that when it is required, its binding to the IRES helps to maintain the appropriate higher-order structure and to reverse distortions caused, for example, by an enlarged A-rich bulge.     

Common conformational changes induced in type 2 picornavirus IRESs by cognate trans-acting factors

Type 2 internal ribosomal entry sites (IRESs) of encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV) and other picornaviruses comprise five major domains H-L. Initiation of translation on these IRESs begins with specific binding of the central domain of initiation factor, eIF4G to the J-K domains, which is stimulated by eIF4A. eIF4G/eIF4A then restructure the region of ribosomal attachment on the IRES and promote recruitment of ribosomal 43S pre-initiation complexes. In addition to canonical translation factors, type 2 IRESs also require IRES trans-acting factors (ITAFs) that are hypothesized to stabilize the optimal IRES conformation that supports efficient ribosomal recruitment: the EMCV IRES is stimulated by pyrimidine tract binding protein (PTB), whereas the FMDV IRES requires PTB and ITAF(45). To test this hypothesis, we assessed the effect of ITAFs on the conformations of EMCV and FMDV IRESs by comparing their influence on hydroxyl radical cleavage of these IRESs from the central domain of eIF4G. The observed changes in cleavage patterns suggest that cognate ITAFs promote similar conformational changes that are consistent with adoption by the IRESs of comparable, more compact structures, in which domain J undergoes local conformational changes and is brought into closer proximity to the base of domain I.     

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.     

The 5' untranslated region of encephalomyocarditis virus contains a sequence for very efficient binding of eukaryotic initiation factor eIF-2/2B.

The mechanism by which internal ribosomal binding on the picornaviral RNA takes place is still not known. An important role has been suggested for eukaryotic initiation factors eIF-4A, eIF-4B, as well as for some not yet defined trans-acting factors like p52 for poliovirus and p58 for encephalomyocarditis virus (EMCV). In this paper we describe the competition between the 5' untranslated region (UTR) of EMCV and globin mRNA for the translational apparatus in rabbit reticulocyte lysates and show that the factor that is competed for is eIF-2/2B. The EMC 5' UTR is a very strong inhibitor of globin synthesis in the rabbit reticulocyte lysate because of a 30-fold higher eIF-2/2B binding capacity. Mutations 100 to 140 nucleotides upstream of the initiation codon led to a decreased efficiency to initiate translation and to a decreased ability to inhibit globin mRNA translation. The results suggest an important role for eIF-2/2B binding in EMC RNA translation and therefore in internal initiation.     

Encephalomyocarditis virus internal ribosomal entry site RNA-protein interactions.

Translational initiation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal entry into the 5' nontranslated region of the EMCV mRNA, rather than by ribosomal scanning. Internal ribosomal binding requires a cis-acting element termed the internal ribosomal entry site (IRES). IRES elements have been proposed to be involved in the translation of picornavirus mRNAs and some cellular mRNAs. Internal ribosome binding likely requires the interaction of trans-acting factors that recognize both the mRNA and the ribosomal complex. Five cellular proteins (p52, p57, p70, p72, and p100) cross-link the EMCV IRES or fragments of the IRES. For one of these proteins, p57, binding to the IRES correlates with translation. Recently, p57 was identified to be very similar, if not identical, to polypyrimidine tract-binding protein. On the basis of cross-linking results with 21 different EMCV IRES fragments and cytoplasmic HeLa extract or rabbit reticulocyte lysate as the source of polypeptides, consensus binding sites for p52, p57, p70, and p100 are proposed. It is suggested that each of these proteins recognizes primarily a structural feature of the RNA rather than a specific sequence.