HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication

EV71 (enterovirus 71) RNA contains an internal ribosomal entry site (IRES) that directs cap-independent initiation of translation. IRES-dependent translation requires the host’s translation initiation factors and IRES-associated trans-acting factors (ITAFs). We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs) from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2) as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication.     

mRNA Decay Factor AUF1 Binds the Internal Ribosomal Entry Site of Enterovirus 71 and Inhibits Virus Replication

AU-rich element binding factor 1 (AUF1) has a role in the replication cycles of different viruses. Here we demonstrate that AUF1 binds the internal ribosome entry site (IRES) of enterovirus 71 (EV71) and negatively regulates IRES-dependent translation. During EV71 infection, AUF1 accumulates in the cytoplasm where viral replication occurs, whereas AUF1 localizes predominantly in the nucleus in mock-infected cells. AUF1 knockdown in infected cells increases IRES activity and synthesis of viral proteins. Taken together, the results suggest that AUF1 interacts with the EV71 IRES to negatively regulate viral translation and replication.     

Neuroattenuation of Vesicular Stomatitis Virus through Picornaviral Internal Ribosome Entry Sites

Vesicular stomatitis virus (VSV) is potent and a highly promising agent for the treatment of cancer. However, translation of VSV oncolytic virotherapy into the clinic is being hindered by its inherent neurotoxicity. It has been demonstrated that selected picornaviral internal ribosome entry site (IRES) elements possess restricted activity in neuronal tissues. We therefore sought to determine whether the picornavirus IRES could be engineered into VSV to attenuate its neuropathogenicity. We have used IRES elements from human rhinovirus type 2 (HRV2) and foot-and-mouth disease virus (FMDV) to control the translation of the matrix gene (M), which plays a major role in VSV virulence. In vitro studies revealed slowed growth kinetics of IRES-controlled VSVs in most of the cell lines tested. However, in vivo studies explicitly demonstrated that IRES elements of HRV2 and FMDV severely attenuated the neurovirulence of VSV without perturbing its oncolytic potency.     

肠道病毒71型基因组中小干扰RNA的靶位点预测

肠道病毒71型（EV71）已经在世界范围内有过十多次大的爆发与流行,近年来EV71病毒的流行在亚洲逐渐呈上升的趋势,但是目前尚无有效的治疗措施,因此迫切需要一种治疗EV71的有效药物。本文采用生物信息学的方法,对人类EV71病毒三个不同株型（SHZH03,SHZH98和MS）RNA序列的局域二级结构进行了预测,并从这些病毒株的基因组中分别得到了长度在21～25nt的小干扰RNA靶序列碱基片段。这一结果将有助于治疗EV71药物的开发研究,对预防和控制EV71的爆发和流行也会有重要意义。     

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…     

Nucleotide Composition of Cellular Internal Ribosome Entry Sites Defines Dependence on NF45 and Predicts a Posttranscriptional Mitotic Regulon

The vast majority of cellular mRNAs initiate their translations through a well-defined mechanism of ribosome recruitment that occurs at the 5′-terminal 7-methylguanosine cap with the help of several canonical protein factors. A subset of cellular and viral mRNAs contain regulatory motifs in their 5′ untranslated regions (UTRs), termed internal ribosome entry sites (IRES), that sidestep this canonical mode of initiation. On cellular mRNAs, this mechanism requires IRES trans-acting protein factors (ITAFs) that facilitate ribosome recruitment downstream of the cap. While several ITAFs and their target mRNAs have been empirically identified, the in silico prediction of targets has proved difficult. Here, we report that a high AU content (>60%) of the IRES-containing 5′ UTRs serves as an excellent predictor of dependence on NF45, a recently identified ITAF. Moreover, we provide evidence that cells deficient in NF45 ITAF activity exhibit reduced IRES-mediated translation of X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 (cIAP1) mRNAs that, in turn, leads to dysregulated expression of their respective targets, survivin and cyclin E. This specific defect in IRES translation explains in part the cytokinesis impairment and senescence-like phenotype observed in HeLa cells expressing NF45 RNA interference (RNAi). This study uncovers a novel role for NF45 in regulating ploidy and highlights the importance of IRES-mediated translation in cellular homeostasis.     

A Predicted Secondary Structural Domain within the Internal Ribosome Entry Site of Echovirus 12 Mediates a Cell-Type-Specific Block to Viral Replication

The enterovirus 5' nontranslated region (NTR) contains an internal ribosome entry site (IRES), which facilitates translation initiation of the viral open reading frame in a 5' (m(7)GpppN) cap-independent manner, and cis-acting signals for positive-strand RNA replication. For several enteroviruses, the 5' NTR has been shown to determine the virulence phenotype. We have constructed a chimera consisting of the putative IRES element from the Travis strain of echovirus 12 (ECV12), a wild-type, relatively nonvirulent human enterovirus, exchanged with the homologous region of a full-length infectious clone of coxsackievirus B3 (CBV3). The resulting chimera, known as ECV12(5'NTR)CBV3, replicates similarly to CBV3 in human and simian cell lines yet, unlike CBV3, is completely restricted for growth on two primary murine cell lines at 37 degrees C. By utilizing a reverse-genetics approach, the growth restriction phenotype was localized to the predicted stem-loop II within the IRES of ECV12. In addition, a revertant of ECV12(5'NTR)CBV3 was isolated which possessed three transition mutations and had restored capability for replication in the utilized murine cell lines. Assays for cardiovirulence indicated that the ECV12 IRES is responsible for a noncardiovirulent phenotype in a murine model for acute myocarditis. The results indicate that the 5' NTRs of ECV12 and CBV3 exhibit variable intracellular requirements for function and serve as secondary determinants of tissue or species tropism.     

Mechanism of Initiation Site Selection Promoted by the Human Rhinovirus 2 Internal Ribosome Entry Site

Translation initiation site usage on the human rhinovirus 2 internal ribosome entry site (IRES) has been examined in a mixed reticulocyte lysate/HeLa cell extract system. There are two relevant AUG triplets, both in a base-paired hairpin structure (domain VI), with one on the 5′ side at nucleotide (nt) 576, base paired with the other at nt 611, which is the initiation site for polyprotein synthesis. A single residue was inserted in the apical loop to put AUG-576 in frame with AUG-611, and in addition another in-frame AUG was introduced at nt 593. When most of the IRES was deleted to generate a monocistronic mRNA, the use of these AUGs conformed to the scanning ribosome model: improving the AUG-576 context increased initiation at this site and decreased initiation at downstream sites, whereas the converse was seen when AUG-576 was mutated to GUA; and AUG-593, when present, took complete precedence over AUG-611. Under IRES-dependent conditions, by contrast, much less initiation occurred at AUG-576 than in a monocistronic mRNA with the same AUG-576 context, mutation of AUG-576 decreased initiation at downstream sites by ∼70%, and introduction of AUG-593 did not completely abrogate initiation at AUG-611, unless the apical base pairing in domain VI was destroyed by point mutations. These results indicate that ribosomes first bind at the AUG-576 site, but instead of initiating there, most of them are transferred to AUG-611, the majority by strictly linear scanning and a substantial minority by direct transfer, which is possibly facilitated by the occasional persistence of base pairing in the apical part of the domain VI stem.Until the recent discovery of animal picornaviruses with internal ribosome entry sites (IRESs) resembling that of hepatitis C virus, most picornavirus IRESs have been classified into two groups (1, 17): type 1 (exemplified by entero- and rhinoviruses) and type 2 (cardio- and aphthoviruses). Primary sequences and especially secondary structures are strongly conserved within each group but there is very little similarity between the two groups apart from an AUG triplet at the 3′ end of the IRES (as defined by deletion analysis), which is preceded by a ∼25 nucleotide (nt) pyrimidine-rich tract (17). In type 2 IRESs, notably encephalomyocarditis virus (EMCV), this AUG triplet is the authentic initiation codon for viral polyprotein synthesis, and the totality of the evidence indicates that all ribosomes bind at, or very close to, this AUG and that all initiate translation at this site (18, 19). The foot-and-mouth disease virus (FMDV), although a type 2 IRES, is not quite so straightforward in that a minority of initiation events occur at the AUG immediately downstream of the oligopyrimidine tract, and the rest occur at the next AUG, 84 nt downstream (3, 45).In contrast, initiation on type 1 IRESs seems much more complicated and rather puzzling. The first puzzling feature is that there is very little, if any, initiation at the AUG just downstream of the oligopyrimidine tract, at nt 586 in poliovirus type 1 (PV-1) (39), and the initiation site for polyprotein synthesis is the next AUG further downstream, at a distance of ∼160 nt in enteroviruses and ∼35 nt in rhinoviruses (17). Nevertheless, AUG-586 is important for efficient initiation at the authentic polyprotein initiation site. Mutation of AUG-586 in a PV-1 infectious clone was found to be quasi-infectious (42), while mutation of the equivalent site in PV-2 conferred a small-plaque phenotype and reduced initiation at the polyprotein initiation site by ∼70% in both in vitro assays and in transfection assays (32, 33, 37).This observation has led to the idea that ribosomes first bind at AUG-586, but instead of initiating at this site, virtually all of them get transferred to the polyprotein initiation site (17). This raises questions as to the nature of the transfer process. Because insertion of an AUG codon between PV-1 nt 586 and the authentic initiation site conferred a small-plaque phenotype and because all large-plaque pseudo-revertants had lost the inserted AUG either by deletion or point mutation (25, 26), linear scanning is likely to be important. However, as the insertion resulted in a small-plaque phenotype rather than lethality, there remains the possibility that some ribosomes were transferred directly without scanning the whole distance. This has also been suggested on the grounds that insertion of AUGs or a hairpin loop between nt 586 and the authentic initiation site of PV-1 did not seem to reduce polyprotein synthesis in vitro as much as might be expected if the authentic initiation site is accessed by strictly linear scanning (8).The final puzzle is that AUG-586 is located in a stem-loop structure, domain VI (Fig. ​(Fig.1A),1A), which is conserved in all entero- and rhinoviruses apart from bovine enterovirus. If the initiating 40S subunits do inspect AUG-586 in some way, albeit an unproductive way, this stem-loop would need to open at least partly, if not completely. This need for domain VI to be opened might be considered an impediment to efficient initiation, and yet its strong conservation suggests the opposite, namely, that it might have a positive effect. Precise deletion of the spacer downstream of AUG-586 in PV-1(Mahoney), so that polyprotein synthesis now started at 586, reduced virus yield by ∼10-fold (39), and in an independent study a deletion that brought the polyprotein initiation site to nt 586 or 580 caused a very similar growth defect in PV-1(Sabin) although the defect was considerably less in a Mahoney background (13, 27). On the other hand, two smaller deletions in PV-1(Sabin) that retained just the whole base-paired domain VI or only its 5′ side, placing the polyprotein initiation site 52 or 31 nt, respectively, downstream of AUG-586, did not confer any significant negative phenotype (13, 27). Taken together, these results would seem to imply that the base pairing in domain VI is neutral to initiation efficiency, but the primary sequence of its 5′ side may confer a moderate positive effect. In this respect it is interesting that bovine enterovirus retains most of the sequence of the 5′ side of domain VI but lacks the complementary sequence of the 3′ side.Open in a separate windowFIG. 1.(A) Sequence and base pairing of IRES domain VI of HRV-2 and PV-1(Mahoney), numbered with respect to the viral genome sequence. (B) Hypothetical model for the opening of HRV-2 domain VI in two stages, showing that in the intermediate state AUG-576 and AUG-611 are both exposed.We have reexamined these issues but in the context of human rhinovirus 2 (HRV-2), mainly because the close proximity of the polyprotein initiation site (at nt 611) to the AUG (at nt 576) just downstream of the oligopyrimidine tract makes the interpretation of results less ambiguous than is the case with enteroviruses. A recent comprehensive sequence comparison of 106 different HRV strains plus 10 field isolates shows that HRV-2 domain VI is typical of the 106 serotypes and the one field isolate that differs in domain VI from its parent strain (35). In 95% of these sequences, the number of residues between the two AUG codons is in the range of 28 to 34 nt (median, 31 nt), with five outliers at 20 or 22 nt. The two AUGs are invariably base paired in a back-to-back configuration (Fig. ​(Fig.1A),1A), and the intervening residues fold into a base-paired structure, usually with a single mismatch (Fig. ​(Fig.1A)1A) or at least one G-U codon at around the mid-point and an apical loop of 3 to 6 residues (depending on the strain). The base-paired stem of enteroviruses is considerably shorter (usually without a mismatch), and the extra length in HRV domain VI generally consists of A-U and U-A pairs (often alternating) in the apical part (Fig. ​(Fig.1A).1A). In 23% of these 107 HRV domain VI sequences, the two AUGs are in the same reading frame, and in 17 (approximately two-thirds) of these there is no in-frame stop codon between them so that any initiation at the upstream AUG would result in synthesis of a VP0 protein (and, hence, also VP4) with an N-terminal extension.We first asked whether AUG-576 in HRV-2 is similar to AUG-586 in PV-1 in that there is very little initiation at this site, and yet AUG-576 is important for efficient initiation at the downstream polyprotein initiation site. We then looked for evidence that the domain VI stem-loop opens and whether all ribosomes access the authentic initiation site (AUG-611) by strictly linear scanning from some upstream site. We conclude that most ribosomes do access AUG-611 in this way, but a significant minority may take a shortcut, which could be facilitated if the apical part of this domain remains closed and base paired, with the single mismatch in the domain VI stem possibly causing the opening of this domain to occur in two stages (Fig. ​(Fig.1B1B).     

A Rat Brain Bicistronic Gene with an Internal Ribosome Entry Site Codes for a Phencyclidine-binding Protein with Cytotoxic Activity

The cloning and characterization of the gene for the fourth subunit of a glutamate-binding protein complex in rat brain synaptic membranes are described. The cloned rat brain cDNA contained two open reading frames (ORFs) encoding 8.9- (PRO1) and 9.5-kDa (PRO2) proteins. The cDNA sequence matched contiguous genomic DNA sequences in rat chromosome 17. Both ORFs were expressed within the structure of a single brain mRNA and antibodies against unique sequences in PRO1- and PRO2-labeled brain neurons in situ, indicative of bicistronic gene expression. Dicistronic vectors in which ORF1 and ORF2 were substituted by either two different fluorescent proteins or two luciferases indicated concurrent, yet independent translation of the two ORFs. Transfection with noncapped mRNA led to cap-independent translation of only ORF2 through an internal ribosome entry sequence preceding ORF2. In vitro or cell expression of the cloned cDNA led to the formation of multimeric protein complexes containing both PRO1 and PRO2. These complexes had low affinity (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801)-sensitive phencyclidine-binding sites. Overexpression of PRO1 and PRO2 in CHO cells, but not neuroblastoma cells, caused cell death within 24–48 h. The cytotoxicity was blocked by concurrent treatment with MK-801 or by two tetrahydroisoquinolines that bind to phencyclidine sites in neuronal membranes. Co-expression of two of the other subunits of the protein complex together with PRO1/PRO2 abrogated the cytotoxic effect without altering PRO1/PRO2 protein levels. Thus, this rare mammalian bicistronic gene coded for two tightly interacting brain proteins forming a low affinity phencyclidine-binding entity in a synaptic membrane complex.A complex of four proteins purified from brain synaptic membranes was shown to have recognition sites for l-glutamate, N-methyl-d-aspartate (NMDA),⁴ and other ligands characteristic of NMDA receptors in brain, including binding sites for the co-agonist glycine, the modulator spermine, the competitive antagonist (+)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), and the ion channel inhibitors thienylcyclohexylpiperidine (TCP) and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801) (1, 2). Reconstitution of the purified complex into planar lipid bilayer membranes leads to the formation of channels with four ion conductance levels upon activation by glutamate or NMDA in the presence of glycine (3). These conductances differ from either the predominant NMDA-activated receptor-ion channels of brain neurons or those formed by reconstitution of the NMDA receptor subunits (4), but are similar to those described for ion channels in rat spinal cord motor neurons (5).The genes for three of the proteins in this complex have been cloned and expressed in heterologous cells (6–10). The gene GRINA for the glutamate-binding protein (GBP) subunit was identified as part of a “learning and memory” module of genes expressed in the entorhinal cortex of the mammalian brain (11), and as the gene responsible for mental retardation and epilepsy in infants with a gene duplication in chromosome 8q24.3 (12). Expression of GRINA in heterologous cells leads to activation of mitogen-activated protein kinases (13), i.e. it may be involved in signal transduction in neurons. Because of the potential role of GBP and of the associated membrane complex in cell signaling, there is a need to fully characterize all components of the complex and reconstitute the intact complex in cells lacking in its expression. The genes for two other components of the complex have been cloned, those for the glycine-binding and CPP-binding proteins. But the gene for the fourth subunit has not yet been cloned.The fourth protein of the complex was identified on SDS-PAGE as an ∼40-kDa protein. To complete the characterization of this complex of proteins, the cDNA for the fourth subunit was cloned, and a corresponding genomic sequence in rat genome was identified. The presence of two open reading frames (ORFs) in the cloned cDNA, the expression of both ORFs in a single mRNA in brain, and the translation in brain of the two proteins coded by the cDNA, led to the investigation of the mechanism of translation of both ORFs. Translation of both ORFs through an internal ribosome entry sequence (IRES) was identified, as was the need for the co-expression of the two proteins to create a functional protein, a phencyclidine-binding protein.     

EV71结构蛋白VP0的表达及抗体的制备

目的:原核表达EV71结构蛋白VP0(VP2+VP4)并制备其多克隆抗体.方法:以肠道病毒71型(EV71)全基因组为模板,设计引物扩增出目的片段VP0,将其克隆至表达载体pET-30a(+),并转化大肠杆菌TG1,筛选出阳性克隆后进行测序.将重组表达载体pET-30a (+)-VP0转入大肠杆菌表达菌株Rosetta中.该重组菌经过IPTG诱导表达并通过SDS-PAGE电泳和Western Blot验证后,有与预期分子量大小一致的蛋白条带,并且主要以包涵体的形式存在.包涵体用6 mol/L盐酸胍溶解,经过Ni-NTA亲和层析法纯化,获得了纯度较高的目的蛋白.将纯化的蛋白免疫新西兰大白兔制备了VP0多克隆抗体,并对该抗体进行了细胞免疫荧光分析.结果:经过大肠杆菌重组表达并纯化得到了纯度较高的VP0蛋白,制备的多克隆抗体经过细胞免疫荧光的验证表明反应性良好.结论:成功地表达VP0蛋白并制备了其多克隆抗体,有利于EV71病毒的检测及下一步对其疫苗的研究.     

Translation of Vascular Endothelial Growth Factor mRNA by Internal Ribosome Entry: Implications for Translation under Hypoxia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic growth factor that promotes compensatory angiogenesis in circumstances of oxygen shortage. The requirement for translational regulation of VEGF is imposed by the cumbersome structure of the 5′ untranslated region (5′UTR), which is incompatible with efficient translation by ribosomal scanning, and by the physiologic requirement for maximal VEGF production under conditions of hypoxia, where overall protein synthesis is compromised. Using bicistronic reporter gene constructs, we show that the 1,014-bp 5′UTR of VEGF contains a functional internal ribosome entry site (IRES). Efficient cap-independent translation is maintained under hypoxia, thereby securing efficient production of VEGF even under unfavorable stress conditions. To identify sequences within the 5′UTR required for maximal IRES activity, deletion mutants were analyzed. Elimination of the majority (851 nucleotides) of internal 5′UTR sequences not only maintained full IRES activity but also generated a significantly more potent IRES. Activity of the 163-bp long “improved” IRES element was abrogated, however, following substitution of a few bases near the 5′ terminus as well as substitutions close to the translation start codon. Both the full-length 5′UTR and its truncated version function as translational enhancers in the context of a monocistronic mRNA.     

Discrimination against RNA Backbones by a ssDNA Binding Protein

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Evidence that Protein Binding Specifies Sites of DNA Demethylation

It has been hypothesized that protein factors may protect CpG islands from methyltransferase during development and that demethylation may involve protein-DNA interactions at demethylated sites. However, direct evidence has been lacking. In this study, demethylation at the EBNA-1 binding sites of the Epstein-Barr virus latent replication origin, oriP, was investigated by using human cells. Several novel findings are discussed. First, there are specific preferential demethylation sites within the oriP region. Second, the DNA sequence of oriP alone is not the target of an active demethylation process. Third, EBNA-1 binding is required for the site-specific demethylation in oriP. Interestingly, CpG sites adjacent to and between the EBNA-1 sites do not become demethylated. Fourth, demethylation of the first DNA strand in oriP at the EBNA-1 binding sites involves a passive (replication-dependent) mechanism. The second-strand demethylation appears to occur through an active mechanism. That is, EBNA-1 protein binding prevents the EBNA-1 binding sites from being remethylated after one round of DNA replication, and it appears that an active demethylase then demethylates these hemimethylated sites. This study provides clear evidence that protein binding specifies sites of DNA demethylation and provides insights into the sequence of steps and the mechanism of demethylation.