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。     

猪瘟病毒囊膜糖蛋白E0的RNA酶活性及其研究进展

猪瘟病毒（CSFV,Classicalswinefevervirus）属于黄病毒科瘟病毒属,同属的成员还有牛病毒性腹泻病毒（BVDV）和羊的边界病病毒（BDV）。猪瘟病毒是一种有囊膜的单股正链RNA病毒,基因组大小约12．3kb,含有一个大的ORF,此ORF编码一个大的多聚蛋白,经宿主和病毒编码蛋白酶的共同作用,在共同翻译中和／或翻译后,将此多聚蛋白加工成病毒的结构蛋白和非结构蛋白．猪瘟病毒基因组的5＇端编码病毒结构蛋白,即衣壳蛋白（C）和三个囊膜糖蛋白（E0、E1、E2）。其中E0和E2能够刺激机体产生中和抗体,并使猪获得免疫力[1,2]．意外发…     

SARS冠状病毒中的核衣壳蛋白

严重急性呼吸综合征(SARS)的元凶是一种新冠状病毒,研究病毒结构蛋白的功能有助于了解病毒的感染、复制和包装等生理过程。其中核衣壳蛋白是SARS冠状病毒中含量最丰富和最保守的结构蛋白,自身聚合后包被病毒RNA基因组形成螺旋状核壳体是SARS冠状病毒成熟的关键步骤;核衣壳蛋白能与病毒或宿主细胞中多种蛋白质相互作用,还能影响宿主细胞的多个通路。因此核衣壳蛋白是一个重要的多功能蛋白质,参与了病毒感染、复制和病毒包装等过程。     

siRNA干扰马赛病毒潜在翻译起始因子ORF314显著抑制病毒复制

马赛病毒是继拟菌病毒之后被发现的第2类阿米巴巨型病毒,拥有庞大的双链DNA基因组。基因组分析显示,马赛病毒上海株(Mar-SH2016)编码4种潜在翻译因子。其中,ORF314基因与真核翻译起始因子eIF2/eIF5的氨基酸同源性约为60%,但其在马赛病毒复制中的作用还有待揭示。本研究设计了3对靶向Mar-SH2016 ORF314基因的siRNA,通过转染宿主卡氏棘阿米巴细胞,分析siRNA对ORF314基因表达水平和病毒复制的影响。用荧光标记的siRNA转染卡氏棘阿米巴细胞,结果显示约1/3阿米巴细胞成功转染。实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)和蛋白免疫印迹实验证实,在Mar-SH2016感染宿主的过程中siRNA能使ORF314基因表达水平显著下降。siRNA干扰后病毒毒力测定结果显示,Mar-SH2016的毒力降低约30%(P<0.01)。结果表明,降低ORF314基因的表达水平能显著抑制Mar-SH2016复制。     

杆状病毒p35蛋白抗凋亡作用及机理

杆状病毒入侵可以诱导昆虫细胞凋亡,作为对抗宿主防御体系的一种策略,病毒自身编码具有抗细胞凋亡活性的蛋白,如p35蛋白和IAP。杆状病毒p35蛋白是一种广泛有效的凋亡抑制因子,能在哺乳纲、昆虫纲和线虫纲中抑制细胞凋亡作用,推测其与细胞凋亡途径上保守的成分Caspase起作用。研究表明,p35蛋白正是通过蛋白酶间的相互作用和p35蛋白的剪切而起作用的。就最近几年在p35蛋白抗凋亡作用机理方面的研究作一综述 。     

VHS蛋白,一种具有mRNA剪切活性的多种功能蛋白质

单纯疱疹病毒UL41基因编码的病毒宿主关闭蛋白(VHS蛋白)是一种核酸酶,具有。RNA剪切活性．可引起宿主细胞蛋白质合成的快速关闭。通过干扰IFN-α／β介导的抗病毒免疫反应、降低宿主细胞MHCI和MHCII类分子的表达、减少免疫系统中病毒抗原的提呈以及抑制宿主先天免疫反应等,VHS蛋白在α疱疹病毒的发病机制和免疫逃避过程中发挥重要作用。     

NDV诱导HeLa细胞发生核糖体应激及对翻译起始复合体的影响

[目的]研究新城疫病毒(Newcastle disease virus,NDV) HBUN/LSRC/F3株(以下简称NDV F3)诱导宫颈癌细胞(HeLa)发生核糖体应激后对eIF2α介导的翻译起始复合体eIF4F的调控作用。[方法]流式细胞术及CCK-8检测细胞凋亡;实时荧光定量PCR (quantitative real-time polymerase chain reaction,qRT-PCR)检测c-Myc基因表达;流式细胞术分析细胞周期;Western blotting技术检测c-Myc、RPS7、Bcl-2、NP、eIF4E及eIF2α蛋白的表达;Western blotting和免疫荧光染色技术检测NP、eIF4E蛋白定位。[结果]与阴性对照组相比,NDV F3抑制HeLa细胞增殖并诱导细胞凋亡。细胞周期中G₀/G₁期出现停滞,c-Myc表达呈时间依赖性抑制,c-Myc与Bcl-2蛋白表达量在0-48 h内逐渐下降,NP蛋白在24 h时生成并逐渐增加,RPS7、eIF4E和eIF2α蛋白含量在0-48 h内呈先增加后降低趋势。Western blotting定位分析及激光共聚焦显微镜结果显示NP蛋白主要存在细胞质中,NP与eIF4E存在共定位现象。[结论]NDV F3诱导HeLa细胞凋亡并引发核糖体应激反应,NP与eIF4E相互作用而抑制eIF2α介导的翻译起始复合体eIF4F形成,阻断其与宿主mRNA之间的联系,同时促进NDV F3 mRNA的翻译,最终造成宿主蛋白翻译抑制。     

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.     

小RNA病毒蛋白与细胞凋亡的关系

很多小RNA病毒科病毒感染宿主细胞后可引发宿主细胞凋亡,这种现象被认为是宿主细胞对抗小RNA病毒侵染的防御机制。凋亡机制可由某些病毒蛋白对细胞产生信号干扰来实现多种凋亡通路。虽然这些凋亡通路的上游事件是不同的,但最后的效应却很一致。此外,一些病毒蛋白具有抑制细胞凋亡的功能,它们能够令感染病毒后的细胞不死亡,形成病毒与宿主细胞共存的持续性感染状态。     

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.     

Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells

Infection of cells by foot-and-mouth disease virus (FMDV) results in the rapid inhibition of host cell protein synthesis. This process is accompanied by the early cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. This cleavage is mediated by the leader (L) protease. Subsequently, as the virus proteins accumulate, secondary cleavages of eIF4G occur. Furthermore, eIF4A (46 kDa), a second component of eIF4F, is also cleaved in these later stages of the infection cycle. The 33-kDa cleavage product of eIF4A has lost a fragment from its N terminus. Transient-expression assays demonstrated that eIF4A was not cleaved in the presence of FMDV L or with the poliovirus 2A protease (which also mediates eIF4G cleavage) but was cleaved when the FMDV 3C protease was expressed. The FMDV 3C protease was also shown in such assays to induce cleavage of eIF4G, resulting in the production of cleavage products different from those generated by the L protease. Consistent with these results, within cells infected with a mutant FMDV lacking the L protease or within cells containing an FMDV replicon lacking L-P1 coding sequences it was again shown that eIF4A and eIF4G were cleaved.     

The binding of foot-and-mouth disease virus leader proteinase to eIF4GI involves conserved ionic interactions

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) initially cleaves itself from the polyprotein. Subsequently, L(pro) cleaves the host proteins eukaryotic initiation factor (eIF) 4GI and 4GII. This prevents protein synthesis from capped cellular mRNAs; the viral RNA is still translated, initiating from an internal ribosome entry site. L(pro) cleaves eIF4GI between residues G674 and R675. We showed previously, however, that L(pro) binds to residues 640-669 of eIF4GI. Binding was substantially improved when the eIF4GI fragment contained the eIF4E binding site and eIF4E was present in the binding assay. L(pro) interacts with eIF4GI via residue C133 and residues 183-195 of the C-terminal extension. This binding domain lies about 25 A from the active site. Here, we examined the binding of L(pro) to eIF4GI fragments generated by in vitro translation to narrow the binding site down to residues 645-657 of human eIF4GI. Comparison of these amino acids with those in human eIF4GII as well as with sequences of eIF4GI from other organisms allowed us to identify two conserved basic residues (K646 and R650). Mutation of these residues was severely detrimental to L(pro) binding. Similarly, comparison of the sequence between residues 183 and 195 of L(pro) with those of other FMDV serotypes and equine rhinitis A virus showed that acidic residues D184 and E186 were highly conserved. Substitution of these residues in L(pro) significantly reduced eIF4GI binding and cleavage without affecting self-processing. Thus, FMDV L(pro) has evolved a domain that specifically recognizes a host cell protein.     

Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro

Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.     

L protease from foot and mouth disease virus confers eIF2-independent translation for mRNAs bearing picornavirus IRES

The leader protease (L^pro) from foot-and-mouth disease virus (FMDV) has the ability to cleave eIF4G, leading to a blockade of cellular protein synthesis. In contrast to previous reports, our present findings demonstrate that FMDV L^pro is able to increase translation driven by FMDV IRES. Additionally, inactivation of eIF2 subsequent to phosphorylation induced by arsenite or thapsigargin in BHK cells blocks protein synthesis directed by FMDV IRES, whereas in the presence of L^pro, significant translation is found under these conditions. This phenomenon was also observed in cell-free systems after induction of eIF2 phosphorylation by addition of poly(I:C).     

Recognition of eukaryotic initiation factor 4G isoforms by picornaviral proteinases

The leader proteinase (L(pro)) of foot and mouth disease virus is a papain-like cysteine proteinase. After processing itself from the polyprotein, L(pro) then cleaves the host protein eukaryotic initiation factor (eIf) 4GI, thus preventing protein synthesis from capped mRNA in the infected cell. We have investigated L(pro) interaction with eIF4GI and its isoform, eIF4GII. L(pro), expressed as a catalytically inactive fusion protein with glutathione S-transferase, binds specifically to eIF4G isomers in rabbit reticulocyte lysates. Deletion and specific mutagenesis were used to map the binding domain on L(pro) to residues 183-195 of the C-terminal extension and to residue Cys(133). These residues of the C-terminal extension and Cys(133) are adjacent in the crystal structure but lie about 25 A from the active site. The region on eIF4GI recognized by the L(pro) C-terminal extension was mapped to residues 640-669 using eIF4GI fragments generated by proteolysis or by in vitro translation. The L(pro) cleavage site at Gly(674) downward arrow Arg(675) was not necessary for binding. Similar experiments with human rhinovirus 2A proteinase (2A(pro)), a chymotrypsin-like cysteine proteinase that also cleaves eIF4G isoforms, revealed that 2A(pro) can also bind to eIF4GI fragments lacking its cleavage site. These experiments strongly suggest a novel interaction between picornaviral proteinases and eIF4G isoforms.     

The foot-and-mouth disease virus leader proteinase gene is not required for viral replication.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase has only two known functions: (i) autocatalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. Cleavage of p220 appears to be important for picornavirus replication, since rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220. To explore the role of L in FMDV replication, we generated synthetic FMDV genomes lacking the L gene and tested their viability in cells. Genomes were constructed with the N-terminal Gly codon of VP4 positioned directly following either the first (Lab) or second (Lb) Met codon of the L protein. Cells transfected with synthetic RNAs lacking L and initiating with the Lab Met codon failed to produce viable virus, but cells transfected with RNAs that utilized the second AUG to drive translation of the viral polyprotein produced viable viruses. These leader-deleted viruses produced plaques on BHK cells that were slightly smaller than those produced by wild-type (WT) virus, grew to slightly lower titers than WT virus in BHK cells, shut off host protein synthesis more slowly than WT virus, and were slightly attenuated in mice. These studies indicate that the L proteinase is not essential for FMDV replication and show that in the cells and animals tested the L gene has a limited effect on virus replication.     

GRP94 (endoplasmin) co-purifies with and is phosphorylated by Golgi apparatus casein kinase

Certain picornaviruses encode proteinases which cleave the translation initiation factor eIF4G, a member of the eIF4F complex which recruits mRNA to the 40S ribosomal subunit during initiation of protein synthesis in eukaryotes. We have compared the efficiency of eIF4G cleavage in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (L^pro) or the human rhinovirus 2A^pro. Under standard translation conditions, L^pro cleaved 50% of eIF4G within 4 min after initiation of protein synthesis, whereas 2A^pro required 15 min. At these times, the molar ratios of proteinase to eIF4G were 1:130 for L^pro and 1:12 for 2A^pro, indicating a much more efficient in vitro cleavage than previously observed. The molar ratios are similar to those observed during viral infection in vivo.     

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.     

Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition.

The leader protease of foot-and-mouth disease virus, as well as cleaving itself from the nascent viral polyprotein, disables host cell protein synthesis by specific proteolysis of a cellular protein: the eukaryotic initiation factor 4G (eIF4G). The crystal structure of the leader protease presented here comprises a globular catalytic domain reminiscent of that of cysteine proteases of the papain superfamily, and a flexible C-terminal extension found intruding into the substrate-binding site of an adjacent molecule. Nevertheless, the relative disposition of this extension and the globular domain to each other supports intramolecular self-processing. The different sequences of the two substrates cleaved during viral replication, the viral polyprotein (at LysLeuLys/GlyAlaGly) and eIF4G (at AsnLeuGly/ArgThrThr), appear to be recognized by distinct features in a narrow, negatively charged groove traversing the active centre. The structure illustrates how the prototype papain fold has been adapted to the requirements of an RNA virus. Thus, the protein scaffold has been reduced to a minimum core domain, with the active site being modified to increase specificity. Furthermore, surface features have been developed which enable C-terminal self-processing from the viral polyprotein.     

Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.