Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins is responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNA translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTR sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNA translation.     

Recruitment of host translation initiation factor eIF4G by the Vaccinia Virus ssDNA-binding protein I3

Poxviruses are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of infected cells within discrete compartments termed viral factories. Recent work has shown that the prototypical poxvirus, Vaccinia Virus (VacV) sequesters components of the eukaryotic translation initiation complex eIF4F within viral factories while also stimulating formation of eIF4F complexes. However, the forces that govern these events remain unknown. Here, we show that maximal eIF4F formation requires viral DNA replication and the formation of viral factories, suggesting that sequestration functions to promote eIF4F assembly, and identify the ssDNA-binding protein, I3 as a viral factor that interacts and co-localizes with the eIF4F scaffold protein, eIF4G. Although it did not adversely affect host or viral protein synthesis, I3 specifically mediated the binding of eIF4G to ssDNA. Combined, our findings offer an explanation for the specific pattern and temporal process of eIF4G redistribution and eIF4F complex assembly within VacV-infected cells.     

Tethering of eIF4G to adenoviral mRNAs by viral 100k protein drives ribosome shunting

Although most mRNAs initiate translation by 5' ribosome scanning, some small fraction of mammalian and viral mRNAs utilize either of two alternate mechanisms, known as internal ribosome entry and ribosome shunting. Ribosome shunting is a poorly understood form of initiation in which 40S ribosome subunits are loaded onto mRNA through interactions with the m7GTP cap, but then bypass large segments of the mRNA as directed by cis-acting RNA shunting elements and trans-acting protein factors. Here, we describe the molecular mechanism by which ribosome shunting occurs with high efficiency on adenovirus late mRNAs. We show that the viral 100k protein possesses a selective binding element for the 5' noncoding region (5'NCR) of viral late mRNAs (known as the tripartite leader), forms a complex with initiation factor eIF4G and poly(A)-binding protein (PABP), and strongly and selectively enhances the level of both factors and 40S ribosome subunits on viral mRNAs in polysomes. Mutational and biochemical studies demonstrate that the ability of 100k protein to bind both the tripartite leader and eIF4G are critical to promote a high level of ribosome shunting. A molecular mechanism for ribosome shunting is described by which enhanced binding of eIF4G and possibly PABP with 100k protein, and simultaneous interaction with the tripartite leader 5'NCR, drives 40S ribosome recruitment and initiation on mRNAs.     

Role of translation initiation factor 4G in lifespan regulation and age-related health

Inhibiting expression of eukaryotic translation initiation factor 4G (eIF4G) arrests normal development but extends lifespan when suppressed during adulthood. In addition to reducing overall translation, inhibition alters the stoichiometry of mRNA translation in favor of genes important for responding to stress and against those associated with growth and reproduction in C. elegans. In humans, aberrant expression of eIF4G is associated with certain forms of cancer and neurodegeneration. Here we review what is known about the roles of eIF4G in molecular, cellular, and organismal contexts. Also discussed are the gaps in understanding of this factor, particularly with regard to the roles of specific forms of expression in individual tissues and the importance of understanding eIF4G for development of potential therapeutic applications.     

多重RT-PCR方法检测甲型流感病毒

目的建立一种快速、敏感、特异的多重RT-PCR,同时检测甲型流感病毒中的3个分型:甲型H1N1流感病毒,季节性H1N1流感病毒,季节性H3N2流感病毒,并将此方法应用到实验室流感病毒核酸检测技术中。方法利用甲型流感病毒3个分型病毒的引物,在同一个RT-PCR反应体系中,对疑似流感咽拭子标本进行检测。结果多重RT-PCR对甲型流感病毒中分型病毒有较高的灵敏度和特异性,可直接从疑似流感标本中同时进行甲型流感病毒分型检测。结论此实验中采用的多重RT-PCR具有与常规RT-PCR一样的特异性和敏感度,而且比普通RT-PCR和病毒分离法更快速,也更简便。     

甲型流感病毒核蛋白基因的克隆表达及纯化

目的　将甲型流感病毒核蛋白(NP)基因克隆到原核表达载体进行可溶性融合表达,制备纯化的病毒核蛋白,为制备甲型流感病毒单克隆抗体提供材料。方法　提取甲型流感病毒RNA ,设计引物,RT PCR扩增NP基因,利用基因工程的手段,将甲型流感病毒的NP基因在大肠埃希菌中进行融合表达,并将表达产物进行亲和层析。结果　成功构建了甲型流感病毒NP基因原核表达载体,经亲和层析制备了较高纯度的目的表达产物。结论　通过合理控制发酵时间、生长温度和诱导物浓度,制备了较为理想的可溶性甲型流感病毒核蛋白。     

环孢霉素A在体内抑制T淋巴细胞的可逆性

为了阐明环孢霉素A(CsA)在体内的作用机制,本文以甲型流感病毒感染的小鼠为模型,在小鼠感染的-1、1、3、5、7、9天于颈背部皮下注射1mg CsA或溶剂。结果表明,CsA处理有利于诱导以肺部高病毒滴度和低病毒清除率为特征的病毒携带状态。用CsA处理的小鼠,在感染后第14天,其脾细胞中Tc细胞的抗病毒杀伤活性和TM细胞诱生淋巴因子(IL-2和IL-3)的能力以及特异性抗体应答均受到强烈的抑制,但在感染后第21天恢复正常。证明CsA在体内能可逆性地抑制淋巴细胞的功能。     

2006年中国流感病毒抗原性及基因特性研究

目的分析2006年中国季节性流感的流行状况，以及病毒的抗原性和基因变异情况。方法对来自流感监测网络的毒株进行单向血凝抑制试验，在此基础上选择不同时间、地点分离的毒株进行血凝素基因的序列测定，然后分析其基因特性。结果2006年我国同时流行A型（H1N1亚型、H3N2亚型）和B型流感病毒。H1N1亚型毒株和B型Victoria系流感病毒为优势毒株。对H1N1亚型毒株的HA1区序列比较发现，2006年分离的毒株与A，湖北洪山／53／2005（H1N1）比较，在192、193、196、198位发生氨基酸替换的毒株．这些位点位于抗原决定簇的B区。H3N2亚型毒株与A，云南，1145／2005（H3N2）比较，在142、144位发生氨基酸替换。我国流行的B型流感毒株无论是Victoria系和Yamagata系毒株的抗原性均没有发生变异，与2005--2006年我国的流行株B／shenzhen／155／2005、B／tianjin／144／2005类似。结论2006年中国流行的H1N1亚型和H3N2亚型流感病毒的抗原性及基因特性已经发生改变；B型流感病毒的抗原性和基因特性没有改变。     

Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation

The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E–Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation.     

Glycogen synthase kinase 3 promotes p53 mRNA translation via phosphorylation of RNPC1

The RNPC1 RNA-binding protein, also called Rbm38, is a target of p53 and a repressor of p53 mRNA translation. Thus, the p53–RNPC1 loop is critical for modulating p53 tumor suppression, but it is not clear how the loop is regulated. Here, we showed that RNPC1 is phosphorylated at Ser195 by glycogen synthase kinase 3 (GSK3). We also showed that GSK3 promotes p53 mRNA translation through phosphorylation of RNPC1. Interestingly, we found that the phosphor-mimetic mutant S195D and the deletion mutant Δ189–204, which lacks the GSK3 phosphorylation site, are unable to repress p53 mRNA translation due to loss of interaction with eukaryotic translation factor eIF4E on p53 mRNA. Additionally, we found that phosphorylated RNPC1, RNPC1-S195D, and RNPC1(Δ189–204) promote p53 mRNA translation through interaction with eukaryotic translation factor eIF4G, which then facilitates the assembly of the eIF4F complex on p53 mRNA. Furthermore, we showed that upon inhibition of the phosphatidylinositol 3-kinase (PI3K)–Akt pathway, GSK3 is activated, leading to increased RNPC1 phosphorylation and increased p53 expression in a RNPC1-dependent manner. Together, we postulate that the p53–RNPC1 loop can be explored to increase or decrease p53 activity for cancer therapy.     

Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNAiMet binding to the ribosome

eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA_i^Met) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE₁) and SE₂, was found to stimulate recruitment of Met-tRNA_i^Met in the ternary complex (TC) with eIF2·GTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI₁ and SI₂, reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA_i^Met in the P site for AUG selection.     

T淋巴细胞在甲型H1N1流感病毒感染中的变化及意义

目的 研究甲型H1N1流行性感冒（流感）患者外周血T淋巴细胞及其激活亚群的变化.方法 用流式细胞仪检测144例甲型HlNl流感患者和41例健康体检者淋巴细胞亚群,对其中83例患者进一步分析治疗前后T淋巴细胞及其激活亚群（HLA-DR＋CD3＋、HLA-DR＋CD4＋和HLA-DR＋CD8＋细胞）.结果 ①与对照组相比,H1N1并发肺炎组和H1N1组淋巴细胞数明显低于对照组,H1N1并发肺炎组淋巴细胞数明显低于H1N1组;H1N1并发肺炎组T淋巴细胞百分比明显低于对照组（P〈0.05）.②治疗前后CD3、CD8细胞百分比和绝对数均差异有统计学意义,治疗前显著降低,CD4细胞数治疗前显著降低.③治疗前后T淋巴细胞激活亚群（HLA-DR＋CD3＋、HLA-DR＋CD4＋和HLA-DR＋CD8＋细胞）百分比差异有统计学意义,治疗前显著降低.结论 测定甲型H1N1流感患者外周血T淋巴细胞及其激活亚群的变化有助于评价甲型H1N1流感患者感染早期的细胞免疫状况,可以作为甲型H1N1流感早期诊断的辅助指标.     

含H5N1-HA基因重组腺病毒疫苗的构建及其诱导免疫应答的初步探讨

目的 构建包含H5N1-HA基因的重组腺病毒疫苗并探讨其免疫效果.方法 用Admax系统构建包含H5N1-HA基因的重组腺病毒疫苗,并用PCR、Western-Blot等方法对重组病毒疫苗进行鉴定;疫苗免疫小鼠后,通过HI实验和ELISPOT实验检测其体液免疫和细胞免疫反应,评价其免疫效果.结果 成功得到了含有H5N1-HA基因的重组腺病毒疫苗;基因表达鉴定表明,HA基因能够在细胞中进行表达;血凝抑制实验结果显示小鼠产生的针对HA抗体滴度在1:320和1:640之间;ELISPOT结果显示实验组和对照组(PBS)相比斑点数量差异有统计学意义(P<0.05),以上免疫结果表明重组腺病毒载体疫苗可以诱导小鼠产生良好的特异性体液和细胞免疫反应.结论 含H5NA-HA的重组腺病毒疫苗可以诱导小鼠产生良好的免疫反应,为研制人禽流感疫苗打下基础.     

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection

Multipeptide constructs, comprising adjacent sequences of the 317–341 intersubunit region of immature influenza A hemagglutinin (H1N1), were designed and the functional properties of these branched peptides were compared to that of the corresponding linear peptides. In vivo studies revealed that the immunogenicity of the peptides was dependent on the presence of the hydrophobic fusion peptide (comprised in FP3), encompassing the N-terminal 1–13 sequence of the HA2 subunit. Antibody and T cell recognition, however, was directed against the 317–329 HA1 sequence, comprised in the P4 peptide. Multiple copies of P4, covalently linked by branched lysine residues, significantly enhanced the efficiency of antibody binding and the capacity of peptides to elicit B- and T-cell responses. A fraction of peptide induced antibodies reacted with immature or with proteolitically cleaved hemagglutinin (HA) molecules pretreated at low pH. Immunization with a multipeptide construct, (P4)₄–FP3, not only resulted in elevated antibody and T cell responses but conferred enhanced protection against lethal A/PR/8/34 (H1N1) infection as compared to its subunit peptides. The beneficial functional properties of this artificial peptide antigen may be acquired by multiple properties including: (i) stabilized peptide conformation which promotes strong, polyvalent binding to both antibodies and MHC class II molecules; (ii) appropriate P4 conformation for antibody recognition stabilized by the covalently coupled fusion peptide, resulting in the production of virus cross reactive antibodies which inhibit the fusion activity of the virus; (iii) activation of peptide specific B cells which potentiate antigen presentation and peptide specific T cell responses; and (iv) generation of helper T cells which secrete lymphokines active in the resolution of infection.     

Differential inhibition of cellular and Sindbis virus translation by brefeldin A

Brefeldin A is a macrolide compound that interferes with the secretory pathway and also affects protein synthesis in mammalian cells. As a result, this antibiotic impedes the maturation of viral glycoproteins of enveloped viruses and viral genome replication in several virus species. In the present work, we show that translation of subgenomic mRNA from Sindbis virus, which in contrast to cellular translation is resistant to brefeldin A after prolonged treatment. The phosphorylation of eIF2alpha as a result of brefeldin A treatment correlates with the inhibition of cellular translation, while late viral protein synthesis is resistant to this phosphorylation. The effect of brefeldin A on Sindbis virus replication was also examined using a Sindbis virus replicon. Although brefeldin A delayed viral RNA synthesis, translation by non-replicative viral RNAs was not affected, reinforcing the idea that brefeldin A delays viral RNA replication, but does not directly affect Sindbis virus protein synthesis.     

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C^pro. Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.     

Molecular changes associated with adaptation of human influenza A virus in embryonated chicken eggs

Failure to isolate A/Fujian/411/2002 (H3N2) in embryonated chicken eggs resulted in its absence from the 2003/2004 vaccine. We analyzed the adaptation of this virus in eggs during serial passages in the amniotic then allantoic cavities. Amniotic passage allowed the virus to grow in the allantoic cavity. During adaptation, 6 amino acid substitutions occurred: 4 in HA (G186V, S219F, V226I, V309I) and 2 in NA (E119Q, Q136K). These substitutions allowed binding to SAalpha2,3Gal- and SAalpha2,6Gal-containing receptors, conferred SAalpha2,3Gal specificity, and preserved antigenicity. Two HA substitutions (G186V, V226I) were sufficient to improve growth. Changing 2 NA residues (E119Q, Q136K) did not improve growth, and adaptation did not result in the HA changes H183L, D188Y, and V226A found by others. These findings suggest that viral adaptation in eggs involves multiple strategies. Vaccine manufacture will benefit from increased understanding of adaptation and strategies to improve human influenza A virus replication in eggs.     

Long term persistence of IgE anti-influenza virus antibodies in pediatric and adult serum post vaccination with influenza virus vaccine

The production of IgE specific to different viruses (HIV-1, Parvovirus B19, Parainfluenza virus, Varicella Zoster Virus), and the ability of IgE anti-HIV-1 to suppress HIV-1 production in vitro, strongly suggest an important role for IgE and/or anti viral specific IgE in viral pathogenesis. Nevertheless, the presence and persistence of IgE anti-Influenza virus antibodies has not been studied. Total serum IgE and specific IgE and IgG anti-Influenza virus antibodies were studied in children (N=3) (m/f 14-16 y/o) and adults (N=3) (m/f, 41-49 y/o) 2-20 months after vaccination with Influenza virus (Flumist^® or Fluzone^®), as well as in non-vaccinated children (N=2). (UniCAP total IgE Fluoroenzymeimmunoassay, ELISA, Immunoblot). We found that serum of vaccinated children and adults contained IgE and IgG anti-Influenza virus antibodies approaching two years post vaccination. Non-vaccinated children did not make either IgE or IgG anti-Influenza antibodies. Similar levels of IL-2, IFN-γ, IL-4, and IL-10 cytokines were detected in serum of vaccinated compared with non vaccinated subjects (p>0.05), as well as between vaccinated adults compared with vaccinated children and non vaccinated subjects (p>0.05). Vaccinated children and adults continue to produce IgE anti-Influenza virus antibodies long term post vaccination. The long term production of IgE anti-Influenza virus antibodies induced by vaccination may contribute to protective immunity against Influenza.     

从人分离出两株甲型流感H9N2亚型毒株内部基因特性的研究

目的 了解2株从人分离出的H9N2亚型毒株内部基因特性，并弄清其来源。方法 用RT-PCR扩增目的基因，用P^CEM-T Vector(美国Promega公司)，4℃过夜连接，重组质粒转入dH5a细菌，筛选阳性菌落，酶切鉴定，送六合通公司自动测序。然后进行进化树分析。结果 2株测定毒株内部基因均为G9基因系，它们相互间除PA基因有差异外，其余5个基因均相同。结论 2株测定毒株的基因组均为G9基因系，它们是由携带不同基因特性H9N2毒株的禽群分别直接感染人，而不是来自同一禽的H9N2亚型流感病毒。