Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.     

The scenario on the origin of translation in the RNA world: in principle of replication parsimony

Background  

It is now believed that in the origin of life, proteins should have been "invented" in an RNA world. However, due to the complexity of a possible RNA-based proto-translation system, this evolving process seems quite complicated and the associated scenario remains very blurry. Considering that RNA can bind amino acids with specificity, it has been reasonably supposed that initial peptides might have been synthesized on "RNA templates" containing multiple amino acid binding sites. This "Direct RNA Template (DRT)" mechanism is attractive because it should be the simplest mechanism for RNA to synthesize peptides, thus very likely to have been adopted initially in the RNA world. Then, how this mechanism could develop into a proto-translation system mechanism is an interesting problem.     

昆虫RNA沉默抗病毒机制研究进展

RNA沉默是昆虫用来抵御病毒入侵的一种普遍而又进化保守的防御机制, 而昆虫病毒也会相应地编码沉默抑制子来破坏宿主的防御功能。本文主要结合果蝇的相关研究成果对昆虫RNA沉默抗病毒机制、 RNA沉默抑制子的作用特征及宿主与病毒的共进化关系做一综述。研究表明, 由小干扰RNA (small interfering RNAs, siRNA)介导的RNA干扰在果蝇抗病毒防御机制中发挥重要作用。果蝇中Dicer-2（Dcr-2）, argonaute-2（AGO2）和双链RNA结合蛋白R2D2是siRNA干扰途径中的3个关键组分, 这3个基因的缺失或突变会显著提高果蝇对RNA病毒的感受性。此外, 果蝇中还鉴定了其他与RNA干扰密切相关的基因, 如vasa intronic gene, aubergine, armitage, rm62 和piwi, 它们在抗病毒感染中同样发挥重要作用。果蝇病毒中已鉴定出3种RNA沉默病毒抑制子（viral suppressors of RNAi, VSRs）, 分别为果蝇FHV病毒沉默抑制子FHV-B2、 果蝇C病毒沉默抑制子DCV-1A及果蝇CrPV病毒沉默抑制子CrPV-1A。FHV-B2和DCV-1A通过与dsRNA或siRNA结合抑制RNA沉默, 而CrPV-1A通过与AGO2结合阻止RISC的形成抑制RNA沉默。在漫长的进化过程中, 病毒和宿主相互博弈, 协同进化。昆虫抗病毒沉默途径中的关键组分通过保持持续和快速进化来对抗高度变异的VSRs。     

野田村病毒RNA的复制机制

野田村病毒科Nodaviradae分为2个属,分别为主要感染昆虫的α野田村病毒属(Alphanodavirus)和主要感染鱼类的β野田村病毒属(Betanodavirus)。野田村病毒的基因组由2条单链正义RNA分子(RNA1和RNA2)所组成,RNA1编码蛋白A,即病毒负责复制病毒两条基因组的依赖RNA的RNA聚合酶催化亚基。RNA2编码衣壳前体蛋白α,此前体蛋白α先组装成原病毒粒子,再经历一次自我催化的成熟切割成2个病毒的衣壳蛋白β和γ,就成了成熟的有感染性的病毒粒子。在RNA复制过程中,从RNA1的3′末端会合成一个不被包装进病毒粒子的亚基因组RNA3。RNA1能在无RNA2的情况下自我复制,并持续地产生亚基因组RNA3,RNA3的合成采取的是提前终止机制。本文还介绍了野田村病毒复制的调节、非结构蛋白的功能和病毒复制在细胞内的定位。     

RNAi的作用机制及其在抗病毒领域的应用

RNA干扰是外源性或内源性双链RNA诱发的mRNA水平上的基因沉默机制。RNA技术具有高效性、特异性。最近将RNA干扰应用于许多病毒性疾病的治疗研究均取得了显著的基因沉默效果,为病毒的预防和治疗开辟了一条新途径。就RNA干扰作用机制及抗病毒效应作一综述。     

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.     

RNA sequence and secondary structural determinants in a minimal viral promoter that directs replicase recognition and initiation of genomic plus-strand RNA synthesis

Viral RNA replication provides a useful system to study the structure and function of RNAs and the mechanism of RNA synthesis from RNA templates. Previously we demonstrated that a 27 nt RNA from brome mosaic virus (BMV) can direct correct initiation of genomic plus-strand RNA synthesis by the BMV replicase. In this study, using biochemical, nuclear magnetic resonance, and thermodynamic analyses, we determined that the secondary structure of this 27 nt RNA can be significantly altered and retain the ability to direct RNA synthesis. In contrast, we find that position-specific changes in the RNA sequence will affect replicase recognition, modulate the polymerization process, and contribute to the differential accumulation of viral RNAs. These functional results are in agreement with the phylogenetic analysis of BMV and related viral sequences and suggest that a similar mechanism of RNA synthesis takes place for members of the alphavirus superfamily.     

Mechanistic analysis of RNA synthesis by RNA-dependent RNA polymerase from two promoters reveals similarities to DNA-dependent RNA polymerase.

The brome mosaic virus (BMV) RNA-dependent RNA polymerase (RdRp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic RNAs in vitro. Although the requirements for (-)-strand RNA synthesis have been characterized previously, the mechanism of subgenomic RNA synthesis has not. Mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for RNA synthesis. Unlike (-)-strand RNA synthesis, which required only a high GTP concentration, subgenomic RNA synthesis required high concentrations of both GTP and UTP. Phylogenetic analysis of the sequences surrounding the initiation sites for subgenomic and genomic (+)-strand RNA synthesis in representative members of the alphavirus-like superfamily revealed that the +1 and +2 positions are highly conserved as a pyrimidine-adenylate. GDP and dinucleotide primers were able to more efficiently stimulate (-)-strand synthesis than subgenomic synthesis under conditions of limiting GTP. Oligonucleotide products of 6-, 7-, and 9-nt were synthesized and released by RdRp in 3-20-fold molar excess to full-length subgenomic RNA. Termination of RNA synthesis by RdRp was not induced by template sequence alone. Our characterization of the stepwise mechanism of subgenomic and (-)-strand RNA synthesis by RdRp permits comparisons to the mechanism of DNA-dependent RNA synthesis.     

RNA干扰技术在水产动物抗病毒和抗寄生虫研究中的应用研究进展

RNA干扰(RNA interference,RNAi)是由双链RNA介导的,抑制目标基因的表达,沉默靶基因的一种转录后基因沉默机制,并且在真核生物中广泛存在。近年来随着水产养殖业的发展壮大,水产动物疾病频繁爆发,给养殖户带来巨大的经济损失。目前,病毒、寄生虫等病原引起的水产动物疾病的致病机制还有待深入研究。RNA干扰技术的出现为水产动物疾病致病机制的研究提供了强有力的工具。主要对RNA干扰的发现、作用机制以及在水产动物抗病毒和抗寄生虫研究中的应用作以综述,并对未来RNAi技术在水产动物疾病防治中的研究和应用进行了展望,旨为水产动物疾病控制提供参考。     

A new aspect of the RNA bacteriophages translation control mechanism.

The polarity effect of the coat protein gene of the ribonucleic acid of RNA bacteriophages on the polymerase gene translation will be taken as the basis of the polymerase translation control mechanism. A further condition for this mechanism discussed in this work is the dependence of the phage RNA replication on host cell translation factors. The ribosome binding sites of the phage RNA play a decisive role to realize the control mechanism coding for definite ribosome binding probabilities. The relation between them quantifies the reached polymerase concentration in the early phase of the development of the RNA bacteriophage system in the infected cell.     

Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a de novo mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function in vitro to repair the 3′ initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the cis-acting elements for RdRP or TNTase activity at the 3′-end of positive or negative strand RNA1. Taken together, our data establish the de novo synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.     

Alternative editing of cytochrome c oxidase III mRNA in trypanosome mitochondria generates protein diversity

Trypanosomes use RNA editing to produce most functional mitochondrial messenger RNA. Precise insertion and deletion of hundreds of uridines is necessary to make full-length cytochrome c oxidase III (COXIII) mRNA. We show that COXIII mRNA can be alternatively edited by a mechanism using an alternative guide RNA to make a stable mRNA. This alternatively edited mRNA is translated to produce a unique protein that fractionates with mitochondrial membranes and colocalizes with mitochondrial proteins in situ. Alternative RNA editing represents a previously unknown mechanism generating protein diversity and, as such, represents an important function for RNA editing.     

转录后基因沉默与植物的病毒抗性

转录后基因沉默(PTGS)是近10年发现的一种生物(特别是真核生物)细胞抵抗外来核酸入侵及保持生物自身基因组完整性的防御机制,特别是与生物的病毒抗性密切相关。PTGS最初在植物内发现,近几年又分别在真菌、动物等生物细胞内发现。经过10年的研究,我们对PTGS的机制和特点有了相当的了解。这不但对深入地了解基因的表达调控机制意义重大,而且还可为人们如何调控和利用PTGS奠定了基础。本文从PTGS的特点、PTGS与病毒抗性、PTGS在真核生物内发生的广泛性等方面进行综述,并对PTGS发生的机制进行了讨论。