Antiviral silencing in animals

RNA silencing or RNA interference (RNAi) refers to the small RNA-guided gene silencing mechanism conserved in a wide range of eukaryotic organisms from plants to mammals. As part of this special issue on the biology, mechanisms and applications of RNAi, here we review the recent advances on defining a role of RNAi in the responses of invertebrate and vertebrate animals to virus infection. Approximately 40 miRNAs and 10 RNAi suppressors encoded by diverse mammalian viruses have been identified. Assays used for the identification of viral suppressors and possible biological functions of both viral miRNAs and suppressors are discussed. We propose that herpes viral miRNAs may act as specificity factors to initiate heterochromatin assembly of the latent viral DNA genome in the nucleus.     

The Ebola virus VP35 protein is a suppressor of RNA silencing

RNA silencing or interference (RNAi) is a gene regulation mechanism in eukaryotes that controls cell differentiation and developmental processes via expression of microRNAs. RNAi also serves as an innate antiviral defence response in plants, nematodes, and insects. This antiviral response is triggered by virus-specific double-stranded RNA molecules (dsRNAs) that are produced during infection. To overcome antiviral RNAi responses, many plant and insect viruses encode RNA silencing suppressors (RSSs) that enable them to replicate at higher titers. Recently, several human viruses were shown to encode RSSs, suggesting that RNAi also serves as an innate defence response in mammals. Here, we demonstrate that the Ebola virus VP35 protein is a suppressor of RNAi in mammalian cells and that its RSS activity is functionally equivalent to that of the HIV-1 Tat protein. We show that VP35 can replace HIV-1 Tat and thereby support the replication of a Tat-minus HIV-1 variant. The VP35 dsRNA-binding domain is required for this RSS activity. Vaccinia virus E3L protein and influenza A virus NS1 protein are also capable of replacing the HIV-1 Tat RSS function. These findings support the hypothesis that RNAi is part of the innate antiviral response in mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian virus replication.     

RNA沉默在植物生物逆境反应中的作用

RNA沉默是真核生物共有的基因表达调节机制和防御机制。在植物RNA沉默中, 一些小RNAs, 如微小 RNAs和小干扰RNAs, 在植物防御病毒、细菌或食草动物的反应中具有重要作用。为了抑制宿主的RNA沉默系统, 植物病毒或细菌进化出了在RNA沉默不同阶段起作用的病毒沉默抑制子或细菌沉默抑制子, 来克服寄主的RNA沉默反应。文章就植物RNA沉默、病毒沉默抑制子、细菌沉默抑制子及其相关防御反应的一些新进展做一概述。     

RNA 沉默的病毒抑制子

RNA 沉默是一种在真核生物体内普遍保守的、通过核酸序列特异性的相互作用来抑制基因表达的调控机制 . RNA 沉默的一种重要生物学效应是防御病毒的侵染,而针对寄主的这种防御机制,许多植物病毒已演化通过编码 RNA 沉默的抑制子来克服这种防御反应 . 目前,已从植物、动物和人类病毒中鉴定了 20 多种 RNA 沉默的抑制子,围绕抑制子的鉴定和作用机理研究已成为病毒学研究的一个热点 . 对 RNA 沉默抑制子的发现、鉴定方法、作用机理及与病毒病症状形成的关系、动物病毒的沉默抑制子等方面的最新进展做了综述,并对沉默抑制子的应用和存在的问题进行了讨论 .     

Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.     

RNA干扰与植物抗病毒

RNA干扰是多种生物体内由双链RNA介导的同源mRNA降解现象,是植物体内天然的抗病毒机制。然而病毒在长期进化过程中也获得了通过编码沉默抑制蛋白来对抗植物体RNAi系统的能力。本文对RNA干扰过程、病毒编码的沉默抑制蛋白及利用干扰技术进行抗病毒基因工程研究进行简要综述。     

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.     

MicroRNAs and human retroviruses

MicroRNAs (miRNAs) are small non-coding RNAs that control a multitude of critical processes in mammalian cells. Increasing evidence has emerged that host miRNAs serve in animal cells to restrict viral infections. In turn, many viruses encode RNA silencing suppressors (RSS) which are employed to moderate the potency of the cell's miRNA selection against viral replication. Some viruses also encode viral miRNAs. In this review, we summarize findings from human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) that illustrate examples of host cell miRNAs that target the viruses, of RSS encoded by viruses, and of host cell miRNA profile changes that are seen in infected cells. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.     

Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway

Cellular RNA interference (RNAi) provides a natural response against viral infection, but some viruses have evolved mechanisms to antagonize this form of antiviral immunity. To determine whether Ebolavirus (EBOV) counters RNAi by encoding suppressors of RNA silencing (SRSs), we screened all EBOV proteins using an RNAi assay initiated by exogenously delivered small interfering RNAs (siRNAs) against either an EBOV or a reporter gene. In addition to viral protein 35 (VP35), we found that VP30 and VP40 independently act as SRSs. Here, we present the molecular mechanisms of VP30 and VP35. VP30 interacts with Dicer independently of siRNA and with one Dicer partner, TRBP, only in the presence of siRNA. VP35 directly interacts with Dicer partners TRBP and PACT in an siRNA-independent fashion and in the absence of effects on interferon (IFN). Taken together, our findings elucidate a new mechanism of RNAi suppression that extends beyond the role of SRSs in double-stranded RNA (dsRNA) binding and IFN antagonism. The presence of three suppressors highlights the relevance of host RNAi-dependent antiviral immunity in EBOV infection and illustrates the importance of RNAi in shaping the evolution of RNA viruses.     

Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan β-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and βC1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and βC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.     

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。     

Suppressors of RNA silencing encoded by plant viruses and their role in viral infections

RNA silencing as a robust host defense mechanism against plant viruses is generally countered by virus-encoded silencing suppressors. This strategy is now increasingly recognized to be used by animal viruses as well. We present here an overview of the common features shared by some of the better studied plant viral silencing suppressors. We then briefly describe the characteristics of the few reported animal viral suppressors, notably their extraordinary ability of cross-kingdom suppression. We next discuss the basis for biased protection of viral RNA and subviral parasites by silencing suppressors, the link between movement and silencing suppression, the influence of temperature on the outcome of viral infection and the effect of viral silencing suppressors on the microRNA pathway.     

Development of plants resistant to tomato geminiviruses using artificial trans‐acting small interfering RNA

RNA interference (RNAi), a conserved RNA‐mediated gene regulatory mechanism in eukaryotes, plays an important role in plant growth and development, and as an antiviral defence system in plants. As a counter‐strategy, plant viruses encode RNAi suppressors to suppress the RNAi pathways and consequently down‐regulate plant defence. In geminiviruses, the proteins AC2, AC4 and AV2 are known to act as RNAi suppressors. In this study, we have designed a gene silencing vector using the features of trans‐acting small interfering RNA (tasiRNA), which is simple and can be used to target multiple genes at a time employing a single‐step cloning procedure. This vector was used to target two RNAi suppressor proteins (AC2 and AC4) of the geminivirus, Tomato leaf curl New Delhi virus (ToLCNDV). The vector containing fragments of ToLCNDV AC2 and AC4 genes, on agro‐infiltration, produced copious quantities of AC2 and AC4 specific siRNA in both tobacco and tomato plants. On challenge inoculation of the agro‐infiltrated plants with ToLCNDV, most plants showed an absence of symptoms and low accumulation of viral DNA. Transgenic tobacco plants were raised using the AC2 and AC4 tasiRNA‐generating constructs, and T₁ plants, obtained from the primary transgenic plants, were tested for resistance separately against ToLCNDV and Tomato leaf curl Gujarat virus. Most plants showed an absence of symptoms and low accumulation of the corresponding viruses, the resistance being generally proportional to the amounts of siRNA produced against AC2 and AC4 genes. This is the first report of the use of artificial tasiRNA to generate resistance against an important plant virus.     

Multiple functions of Rice dwarf phytoreovirus Pns10 in suppressing systemic RNA silencing

RNA silencing is a potent mechanism of antiviral defense response in plants and other organisms. For counterdefense, viruses have evolved a variety of suppressors of RNA silencing (VSRs) that can inhibit distinct steps of a silencing pathway. We previously identified Pns10 encoded by Rice dwarf phytoreovirus (RDV) as a VSR, the first of its kind from double-stranded RNA (dsRNA) viruses. In this study we investigated the mechanisms of Pns10 function in suppressing systemic RNA silencing in the widely used Nicotiana benthamiana model plant. We report that Pns10 suppresses local and systemic RNA silencing triggered by sense mRNA, enhances viral replication and/or viral RNA stability in inoculated leaves, accelerates the systemic spread of viral infection, and enables viral invasion of shoot apices. Mechanistically, Pns10 interferes with the perception of silencing signals in recipient tissues, binds double-stranded small interfering RNA (siRNAs) with two-nucleotide 3' overhangs, and causes the downregulated expression of RDR6. These results significantly deepen our mechanistic understanding of the VSR functions encoded by a dsRNA virus and contribute additional evidence that binding siRNAs and interfering with RDR6 expression are broad mechanisms of VSR functions encoded by diverse groups of viruses.     

Multiple domains of the tobacco mosaic virus p126 protein can independently suppress local and systemic RNA silencing

Small RNA-mediated RNA silencing is a widespread antiviral mechanism in plants and other organisms. Many viruses encode suppressors of RNA silencing for counter-defense. The p126 protein encoded by Tobacco mosaic virus (TMV) has been reported to be a suppressor of RNA silencing but the mechanism of its function remains unclear. This protein is unique among the known plant viral silencing suppressors because of its large size and multiple domains. Here, we report that the methyltransferase, helicase, and nonconserved region II (NONII) of p126 each has silencing-suppressor function. The silencing-suppression activities of methyltransferase and helicase can be uncoupled from their enzyme activities. Specific amino acids in NONII previously shown to be crucial for viral accumulation and symptom development are also crucial for silencing suppression. These results suggest that some viral proteins have evolved to possess modular structural domains that can independently interfere with host silencing, and that this may be an effective mechanism of increasing the robustness of a virus.