Structural insights into the arms race between host and virus along RNA silencing pathways in Arabidopsis thaliana

RNA silencing refers to a conserved sequence‐specific gene‐regulation mechanism mediated by small RNA molecules. In plants, microRNA (miRNA) and small interfering RNA (siRNA) represent two major types of small RNA molecules which play pivotal roles in plant developmental control and antiviral defences. To escape these plant defences, plant viruses have encoded a vast array of viral suppressors of RNA silencing (VSRs) to attack the host antiviral silencing pathway by interfering with small RNA processing, RNA‐induced silencing complex (RISC) assembly, viral mRNA cleavage etc. Transgenic plants expressing distinct VSRs often show developmental aberrations that resemble the phenotype of miRNA‐deficient mutants, implying a potential intrinsic link between VSRs and the miRNA pathway (at least in Arabidopsis thaliana) even though their pathogenic mechanisms remain largely unknown. In this review, we summarise our current structural understandings of the arms race between the host and virus along the RNA silencing pathway in A. thaliana by focusing on several important ribonucleoprotein (RNP) structures involved in RNA silencing and unique structural features adopted by VSRs.     

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants.     

Polerovirus protein P0 prevents the assembly of small RNA‐containing RISC complexes and leads to degradation of ARGONAUTE1

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double‐stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein–protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA‐induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre‐programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high‐molecular‐weight complex, suggesting the existence of a multi‐protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA–RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co‐expressed AGO1 in both the presence and absence of P0.     

The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways

One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3' overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised.     

siRNA与miRNA在生物基因调控中的沉默机制比较

siRNA和miRNA的沉默机制是生物基因调控的重要手段之一. 小干扰RNA（small interfering RNA,siRNA）是RNA干扰的引发物,激发与之互补的目标mRNA沉默. 非编码RNA中的微小RNA（microRNA,miRNA）,能够识别特定的目标mRNA,通过与mRNAs的3′ 非翻译区结合,影响该目标蛋白的翻译水平. siRNA和miRNA的基因调控机制对生物学研究及疾病的病因和治疗等有直接影响. 本文主要对siRNAs和miRNAs的生物起源及沉默机制进行比较性论述：提出Dicers酶蛋白、Ago蛋白以及20 nt~25 nt的双链RNAs的 3类大分子是RNA沉默的特征结构,并进行了说明性论述|总结性叙述了siRNA和miRNA的2类小分子经典沉默机制,并提出其异同点. 最后,本文根据近期研究进展,对siRNA和miRNA的生物起源及沉默机制提出了新的疑问.     

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.     

Sequence homology required by human immunodeficiency virus type 1 to escape from short interfering RNAs

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5' end or the 3' end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5' and 3' ends are partially tolerated.     

Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level

Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the protoplasts could be induced rapidly and specifically by co-transfection with the reporter gene and various silencing inducers [i.e. siRNA, double-stranded RNA (dsRNA) or plasmid encoding dsRNA]. Using this system, we uncovered novel roles of some viral suppressors. Notably, the Cucumber mosaic virus 2b protein, shown previously to function predominantly by preventing the long-distance transmission of systemic silencing signals, was a very strong silencing suppressor in the protoplasts. Some suppressors thought to interfere with upstream steps of siRNA production appeared to also act downstream. Therefore, a viral suppressor can affect multiple steps of the RNA silencing pathway. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells.     

MicroRNA-like antivirals

Employing engineered DNA templates to express antiviral microRNA (miRNA) sequences has considerable therapeutic potential. The durable silencing that may be achieved with these RNAi activators is valuable to counter chronic viral infections, such as those caused by HIV-1, hepatitis B, hepatitis C and dengue viruses. Early use of expressed antiviral miRNAs entailed generation of cassettes containing Pol III promoters (e.g. U6 and H1) that transcribe virus-targeting short hairpin RNA mimics of precursor miRNAs. Virus escape from single gene silencing elements prompted later development of combinatorial antiviral miRNA expression cassettes that form multitargeting siRNAs from transcribed long hairpin RNA and polycistronic primary miRNA sequences. Weaker Pol III and Pol II promoters have also been employed to control production of antiviral miRNA mimics, improve dose regulation and address concerns about toxicity caused by saturation of the endogenous miRNA pathway. Efficient delivery of expressed antiviral sequences remains challenging and utilizing viral vectors, which include recombinant adenoviruses, adeno-associated viruses and lentiviruses, has been favored. Investigations using recombinant lentiviruses to transduce CD34+ hematological precursor cells with expressed HIV-1 gene silencers are at advanced stages and show promise in preclinical and clinical trials. Although the use of expressed antiviral miRNA sequences to treat viral infections is encouraging, eventual therapeutic application will be dependent on rigorously proving their safety, efficient delivery to target tissues and uncomplicated large scale preparation of vector formulations. This article is part of a special issue entitled: MicroRNAs in viral gene regulation.     

Maintaining inhibition: siRNA double expression vectors against coxsackieviral RNAs

The potential of RNA interference (RNAi) to inhibit virus propagation has been well established in recent years. In several studies, however, emergence of viral escape mutants after prolonged exposure to RNAi has been observed, raising a major hurdle for a possible therapeutic application of this strategy. Here, we report the design and characterisation of a vector that allows the simultaneous expression of two short hairpin RNAs (shRNAs), thereby maintaining high silencing activity even against a viral RNA bearing mutations in one of the target sites. Two short interfering RNAs (siRNAs) against the 3D-RNA dependent RNA polymerase of coxsackievirus B3 were identified that displayed efficient inhibition of virus propagation in HeLa cells and reduced the virus titre by up to 90%. We generated two expression vectors encoding these newly identified siRNAs and evaluated their silencing efficiency against the target gene in a reporter assay. Viral escape was then simulated by introducing a point mutation into either of the target sites. This substitution led to complete abrogation of silencing by the respective vector. To bypass this blockade of silencing, an siRNA double expression vector (SiDEx) was constructed to achieve simultaneous expression of both siRNAs from one plasmid. The silencing efficiency of both siRNAs generated by SiDEx was comparable to that of the individual mono-expression vectors. In contrast to the conventional expression vectors, SiDEx displayed substantial gene regulation also of the mutated target RNA. As our approach of expressing various shRNAs from one vector is based on a simple and universally applicable cloning strategy, SiDEx may be a helpful tool to achieve sustained silencing of viruses, ultimately reducing the risk of emergence of viable mutants. An additional application of SiDEx vectors will be the simultaneous knockdown of two targeted genes for functional studies.     

A silencing safeguard: links between RNA silencing and mRNA processing in Arabidopsis

RNA silencing is a genome defense mechanism used by many eukaryotic organisms to fight viruses and to control transposable elements. Work by Gregory et al. on Arabidopsis thaliana (in this issue of Developmental Cell) revealed a mechanism whereby the plant protects its endogenous messenger RNAs from undergoing RNA silencing and uncovered an unexpected role of the cap-binding protein ABH1 in miRNA biogenesis.     

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。