Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector

Background

Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the green rice leafhopper, Nephotettix cincticeps, in a persistent-propagative manner. Plant reoviruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. Nonstructural protein Pns4 of RDV, a phosphoprotein, is localized around the viroplasm matrix and forms minitubules in insect vector cells. However, the functional role of Pns4 minitubules during viral infection in insect vector is still unknown yet.

Methods

RNA interference (RNAi) system targeting Pns4 gene of RDV was conducted. Double-stranded RNA (dsRNA) specific for Pns4 gene was synthesized in vitro, and introduced into cultured leafhopper cells by transfection or into insect body by microinjection. The effects of the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene on viral replication and spread in cultured cells and insect vector were analyzed using immunofluorescence, western blotting or RT-PCR assays.

Results

In cultured leafhopper cells, the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene strongly inhibited the formation of minitubules, preventing the accumulation of viroplasms and efficient viral infection in insect vector cells. RNAi induced by microinjection of dsRNA from Pns4 gene significantly reduced the viruliferous rate of N. cincticeps. Furthermore, it also strongly inhibited the formation of minitubules and viroplasms, preventing efficient viral spread from the initially infected site in the filter chamber of intact insect vector.

Conclusions

Pns4 of RDV is essential for viral infection and replication in insect vector. It may directly participate in the functional role of viroplasm for viral replication and assembly of progeny virions during viral infection in leafhopper vector.

     

Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic rice plants to the virus

The non-structural protein Pns12 of Rice dwarf virus is one of the early proteins expressed in cultured insect cells, and it is one of 12 proteins that initiate the formation of the viroplasm, the putative site of viral replication. Pns4 is also a non-structural protein, visible as minitubules after nucleation of the viroplasm. We introduced Pns12- and Pns4-specific RNA interference (RNAi) constructs into rice plants. The resultant transgenic plants accumulated short interfering RNAs specific to the constructs. The progeny of rice plants with Pns12-specific RNAi constructs, after self-fertilization, were strongly resistant to viral infection. By contrast, resistance was less apparent in the case of rice plants with Pns4-specific RNAi constructs, and delayed symptoms appeared in some plants of each line. Our results suggest that interference with the expression of a protein that is critical for viral replication, such as the viroplasm matrix protein Pns12, might be a practical and effective way to control viral infection in crop plants.     

Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect

Rice dwarf virus (RDV) replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi) induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.     

New Model for the Genesis and Maturation of Viroplasms Induced by Fijiviruses in Insect Vector Cells

Plant reoviruses are thought to replicate and assemble within cytoplasmic, nonmembranous structures called viroplasms. Here, we established continuous cell cultures of the white-backed planthopper (Sogatella furcifera Horváth) to investigate the mechanisms for the genesis and maturation of the viroplasm induced by Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus in the family Reoviridae, during infection of its insect vector. Electron and confocal microscopy revealed that the viroplasm consisted of a granular region, where viral RNAs and nonstructural proteins P6 and P9-1 accumulated, and a filamentous region, where viral RNAs, progeny cores, viral particles, as well as nonstructural proteins P5 and P6 accumulated. Our results suggested that the filamentous viroplasm matrix was the site for the assembly of progeny virions. Because viral RNAs were produced by assembled core particles within the filamentous viroplasm matrix, we propose that these viral RNAs might be transported to the granular viroplasm matrix. P5 formed filamentous inclusions and P9-1 formed granular inclusions in the absence of viral infection, suggesting that the filamentous and granular viroplasm matrices were formed primarily by P5 and P9-1, respectively. P6 was apparently recruited in the whole viroplasm matrix by direct interaction with P9-1 and P5. Thus, the present results suggested that P5, P6, and P9-1 are collectively required for the genesis and maturation of the filamentous and granular viroplasm matrix induced by SRBSDV infection. Based on these results, we propose a new model to explain the genesis and maturation of the viroplasms induced by fijiviruses in insect vector cells.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti.     

Using RNA interference to develop dengue virus resistance in genetically modified Aedes aegypti

Diseases caused by arthropod-borne viruses are significant public health problems, and novel methods are needed to control pathogen transmission. We hypothesize that genetic manipulation of Aedes aegypti mosquitoes can profoundly and permanently reduce vector competence and subsequent transmission of dengue viruses (DENV) to human hosts. We have identified RNA interference (RNAi) as a potential anti-viral, intracellular pathway in the vector that can be triggered by expression of virus-specific, double stranded RNAs (dsRNAs) to reduce vector competence to DENV. We identified DENV-derived RNA segments using recombinant Sindbis viruses to trigger RNAi, that when expressed in mosquitoes ablate homologous DENV replication and transmission. We also demonstrated that heritable expression of DENV-derived dsRNA in cultured mosquito cells can silence virus replication. We now have developed a number of transgenic mosquito lines that transcribe the effector dsRNA from constitutive promoters such as immediate early 1 (baculovirus) and polyubiquitin (Drosophila melanogaster). We have detected DENV-specific small interfering RNAs, the hallmark of RNAi, in at least one of these lines. Surprisingly, none of these lines expressed dsRNA in relevant tissues (e.g., midguts) that will ultimately affect transmission. A major challenge now is to express the effector dsRNA from tissue-specific promoters to allow RNAi to silence virus replication at critical sites in the vector such as midguts and salivary glands. If successful, this strategy has the advantage of harnessing a naturally occurring vector response to block DENV infection in a mosquito vector and profoundly affect virus transmission.     

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.     

Inhibition of herpesvirus-6B RNA replication by short interference RNAs

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.     

Metabolic engineering of the baculovirus-expression system via inverse "shotgun" genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity

RNA interference (RNAi) is as powerful tool for characterizing gene function in eukaryotic organisms and cultured cell lines. Its use in metabolic engineering has been limited and few reports have targeted protein expression systems to increase yield. In this work, we examine the use of in vitro synthesized double stranded RNA (dsRNA) in the baculovirus expression vector system (BEVS), using commercially relevant cultured cells (Spodoptera frugiperda, Sf-9) and larvae (Trichoplusia ni) as hosts. First, we employed an inverse "shotgun" genomic analysis to "find" an array of 16 putative insect gene targets. We then synthesized dsRNA in vitro targeting these genes and investigated the effects of injected dsRNA on larval growth, development, and product yield. Growth and development was at times stunted and in several cases, the effects were lethal. However, dsRNA targeting an acidic juvenile hormone-suppressible protein (AJHSP1), and translational elongation factor 2 (Ef-2) resulted in significantly increased yield of model product, GFP. Next, we targeted known genes, v-cath and apoptosis inducer, sf-caspase 1, in cultured Sf-9 cells. We confirm RNAi-mediated sf-caspase 1 suppression in Sf-9 cells, but not in baculovirus-infected cells, likely due to the overriding effects of inhibitor of apoptosis protein, p35. We also demonstrate suppression of v-cath in infected cells, which leads to a approximately 3-fold increase in product yield. Overall, our results support the application of RNAi in metabolic engineering, specifically for enhancing protein productivity in the baculovirus expression vector system.     

Chitosan and its quaternized derivative as effective long dsRNA carriers targeting shrimp virus in Spodoptera frugiperda 9 cells

RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(?), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.     

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.     

RNA 干扰技术抑制病毒复制的研究进展

RNA干扰是指dsRNA抑制细胞内同源基因表达的现象,RNA干扰现象自发现以来在短短的几年里,已成功地用于不同种属的生物研究。dsRNA不仅参与内源基因表达调控,而且能够抑制宿主内病原微生物基因的表达,参与构筑生物体的防御机制。近年来,运用RNAi技术在哺乳动物中的研究不断深入,尤其是抑制病毒复制的研究成果令人欣喜,这为人类动物抗病毒治疗提供了新的思路。     

Rotavirus replication: plus-sense templates for double-stranded RNA synthesis are made in viroplasms

Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus.