Production Of Double-stranded RNA Research Articles

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Abstract NG04: Targeting DHX9 to trigger viral mimicry and immunotherapy responsiveness in small cell lung cancer

Proteolytic control of the RNA silencing machinery.

Studies in plants were often pioneering in the field of RNA silencing and revealed a broad range of small RNA (sRNA) categories. When associated with ARGONAUTE (AGO) proteins, sRNAs play important functions in development, genome integrity, stress responses, and antiviral immunity. Today, most of the protein factors required for the biogenesis of sRNA classes, their amplification through the production of double-stranded RNA, and their function in transcriptional and post-transcriptional regulation have been identified. Nevertheless, and despite the importance of RNA silencing, we still know very little about their post-translational regulation. This is in stark contrast with studies in metazoans, where different modifications such as prolyl hydroxylation, phosphorylation, sumoylation, ubiquitylation, and others have been reported to alter the activity and stability of key factors, such as AGO proteins. Here, we review current knowledge of how key components of the RNA silencing machinery in plants are regulated during development and by microbial hijacking of endogenous proteases.

Production of Double-Stranded RNA in Planta by a Potato Mop-Top Virus (PMTV)-Based Vector for Inducing Gene Silencing.

RNA silencing (also known as gene silencing) is an evolutionary conserved mechanism that is involved in regulating gene expression, suppressing mobile elements, and defensing virus infection. RNA silencing is triggered by double-stranded RNA via Dicer or Dicer-like riboendonucleases. DsRNAs are also the replication intermediates of all RNA viruses; as a result, plant RNA viruses are ideal candidates to induce RNA silencing. A large body of plant viruses have been modified into vectors for RNA silencing in varied plant species. Here, we described a simple, time-saving, and operable system for gene function and genetic breeding study of potato and Nicotiana benthamiana using a potato mop-top (MPTV)-based vector.

Production of Double-Stranded RNA Using the Prokaryotic Promoter-Mediated Bidirectional Transcription.

Large-scale and cost-less production of double-stranded RNA (dsRNA) is the basis for the widespread application of dsRNA in agriculture. Bidirectional transcription of target sequence in RNase III-deficient Escherichia coli strain HT115 (DE3) is an efficient way to produce large amounts of dsRNA. Here, we present a detailed method for the production of dsRNA by bidirectional transcription in E. coli from vector construction, induction of expression by isopropylthio-β-galactoside (IPTG), and purification of dsRNA from E. coli.

Bacteria-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management.

Since the discovery of the RNA interference (RNAi) mechanism, it has been widely used in the fields of gene function, biomedicine, and crop pest control. In the direction of agricultural application, this technology is highly expected, especially in the field of pest control, which is called "the third revolution in the history of pesticides". Currently, RNA biopesticides are developing rapidly all over the world. A genetically modified product (MON87411) has been approved for marketing, and a large number of agricultural companies are developing products based on direct spraying RNA biopesticides and submitting them for regulatory approval. The biggest problem that has emerged for spray RNA biopesticides is the technology for large-scale and low-cost production of dsRNA. At present, the bacterial fermentation production technology can realize large-scale dsRNA production with a yield of 4.23~182mg/L bacterial solution. This article describes the experimental protocol for dsRNA production technology based on bacterial fermentation.

Using dsRNA Targeting unc-22 to Demonstrate Gene Knockdown Phenotypes in C.elegans in the Undergraduate Biology Lab Setting

The discovery of RNA interference (RNAi) and its role in gene silencing has revolutionized the study of genetics. In this study, we utilized Caenorhabditis elegans (C. elegans) to demonstrate the effects of RNAi in an undergraduate laboratory setting. Specifically, we targeted the unc-22 gene, responsible for coding the 'twitchin' protein which affects muscle coordination in C. elegans. By employing the L4440 vector, we inserted the unc22 gene into E.coli cells and then induced the production of double-stranded RNA (dsRNA) targeting unc-22 using IPTG. Feeding the C. elegans with these dsRNA-expressed E.coli resulted in a 'twitcher' phenotype, indicative of the unc-22 gene knockdown. This streamlined project offered students not only hands-on molecular experimentation but also experience in data analysis of worm movement. Moreover, through gel electrophoresis and bacterial transformation, students observed the nuances of RNAi machinery in action. The study reaffirms the importance of using C. elegans as a model organism for genetics and evolution studies and offers an experiential learning platform for students to understand the fundamental mechanisms of gene silencing.

Vaccines' New Era-RNA Vaccine.

RNA vaccines, including conventional messenger RNA (mRNA) vaccines, circular RNA (circRNA) vaccines, and self-amplifying RNA (saRNA) vaccines, have ushered in a promising future and revolutionized vaccine development. The success of mRNA vaccines in combating the COVID-19 pandemic caused by the SARS-CoV-2 virus that emerged in 2019 has highlighted the potential of RNA vaccines. These vaccines possess several advantages, such as high efficacy, adaptability, simplicity in antigen design, and the ability to induce both humoral and cellular immunity. They also offer rapid and cost-effective manufacturing, flexibility to target emerging or mutant pathogens and a potential approach for clearing immunotolerant microbes by targeting bacterial or parasitic survival mechanisms. The self-adjuvant effect of mRNA-lipid nanoparticle (LNP) formulations or circular RNA further enhances the potential of RNA vaccines. However, some challenges need to be addressed. These include the technology's immaturity, high research expenses, limited duration of antibody response, mRNA instability, low efficiency of circRNA cyclization, and the production of double-stranded RNA as a side product. These factors hinder the widespread adoption and utilization of RNA vaccines, particularly in developing countries. This review provides a comprehensive overview of mRNA, circRNA, and saRNA vaccines for infectious diseases while also discussing their development, current applications, and challenges.

Seneca Valley Virus Induces DHX30 Cleavage to Antagonize Its Antiviral Effects.

Seneca Valley virus (SVV) is a new pathogen associated with porcine idiopathic vesicular disease (PIVD) in recent years. However, SVV-host interaction is still unclear. In this study, through LC-MS/MS analysis and coimmunoprecipitation analysis, DHX30 was identified as a 3Cpro-interacting protein. 3Cpro mediated the cleavage of DHX30 at a specific site, which depends on its protease activity. Further study showed that DHX30 was an intrinsic antiviral factor against SVV that was dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of viral infection. RIP-seq showed comparatively higher coverage depth at SVV 5'UTR, but the distribution across SVV RNA suggested that the interaction had low specificity. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. Interestingly, DHX30 was determined to interact with 3D in an SVV RNA-dependent manner. Thus, DHX30 negatively regulated SVV propagation by blocking viral RNA synthesis, presumably by participating in the viral replication complex. IMPORTANCE DHX30, an RNA helicase, is identified as a 3Cpro-interacting protein regulating Seneca Valley virus (SVV) replication dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of virus infection. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. In addition, 3Cpro abolished DHX30 antiviral effects by inducing DHX30 cleavage. Thus, DHX30 is an intrinsic antiviral factor that inhibits SVV replication.

Selected Articles from This Issue

Ewing sarcoma is characterized by oncogenic gene fusions and elevated lysine-specific demethylase-1 (LSD1) expression. While Theisen and colleagues previously devised a small molecule LSD1 inhibitor that demonstrates therapeutic efficacy against Ewing sarcoma — SP-2509 — treatment with SP-2509 alone potentiates drug resistance. In this study, Tokarsky et al., sought to elucidate molecular mechanisms underlying SP-2509 resistance by using a CRISPR–Cas9 loss-of-function screen in SP-2509–sensitive Ewing sarcoma cell lines. Results from the screen demonstrated that abrogating expression of mitochondrial ribosomal protein L45 (MRPL45) as well as members of electron transport chain complexes III (CIII) and IV (CIV) reduces SP-2509 responsiveness. Accordingly, CIII inhibitors and targeted removal of genes encoding CIII constituents ubiquinone-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 (UQCRFS1) and cytochrome c1 (CYC1) using CRISPR–Cas9 induces SP-2509 resistance in Ewing sarcoma cell lines. Silencing UQCRFS1, CYC1, or MRPL45 expression diminishes transcriptional responses to SP-2509, indicating that mitochondrial function contributes to transcriptional programs underlying SP-2509 cytotoxicity. Overall, this study implicates mitochondrial dysfunction as a mechanism facilitating SP-2509 resistance, providing a target that may augment SP-2509 efficacy.Germline BRCA1/2 mutations increase the risk of multiple cancers that exhibit homologous recombination deficiency (HRD). BRCA1/2 mutations and subsequent HRD are associated with a number of genomic mutational signatures that have been previously described and seem to represent use of backup DNA repair pathways. These signatures, such as HRDetect and others, have been quantified and used to inform prognoses and therapeutic strategies. In this study, Moore and colleagues used current pre-clinical knowledge about polymerase theta-mediated end joining (TMEJ) and the genomic scars most typical of this backup repair pathway to determine if TMEJ signatures have prognostic value in advanced ovarian cancer. The authors found that both a TMEJ deletion signature with 4 or more base pair stretches of microhomology (TMEJ4) and TMEJ-associated templated insertions (TINS) are enriched in tumors with BRCA1/2 mutations. The authors also discovered that patients displaying high HRDetect and TINS signatures survive significantly longer than patients displaying one or neither signature. Taken together, this study highlights the potential utility of TMEJ mutational signatures in advanced ovarian cancer prognosis.Immune checkpoint inhibitors (ICI) are ineffective in pancreatic cancer, rendering combination treatments capable of augmenting ICI imperative. Radiation and inhibition of DNA damage response proteins, such as DNA-dependent protein kinase (DNA-PK), can activate antitumor immunity. However, whether and how DNA-PK inhibition mediates antitumor immunity in typically immunology “cold” cancers, including pancreatic cancer, is unknown. In their study, Wang and colleagues used radiation and DNA-PK inhibitor M3814 in mouse pancreatic cancer models to test whether the combination regimen can enhance antitumor immunity. The authors found that radiation-M3814 treatment induces type I interferon (T1IFN) signaling and slows tumor growth uniquely in mice with intact immune systems. The authors demonstrated that radiation and M3814-mediated T1IFN signaling is dependent on RNA polymerase III (POL III)-retinoic acid-inducible gene I (RIG-I)-mitochondrial anti-viral-signaling protein (MAVS) signaling, presumably through production and recognition of double-stranded RNA. Furthermore, T1IFN signaling induces tumor PD-L1 expression, and radiation-M3814 treatment augments anti-PD-L1 efficacy in vivo. In sum, this study elucidates a novel mechanism by which ICI efficacy may be enhanced in typically immunologically “cold” pancreatic cancers.Increased MYC stabilization is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), but molecular mechanisms underlying MYC stabilization in T-ALL are incompletely understood. To study effects of two MYC mutations — T58A and S62A — on MYC stabilization and MYC-induced pathophysiology in T-ALL, Daniel and colleagues generated T-cell–specific MycWT, MycT58A, and MycS62A knock-in mouse models in which endogenous wild-type Myc can be maintained or eliminated. The authors observed that MycT58A induces heightened ex vivo T-cell proliferation and thymic mass relative to MycS62A, and that MycT58A causes T-cell lymphomagenesis more rapidly than does MycS62A. Nonetheless, MycS62A does induce clonal T-cell lymphomas that resemble T-ALL and, unlike MycT58A, causes non-transgene driven, incidental thymic B-cell lymphomas with splenomegaly. Differential phenotypes engendered by MycT58A and MycS62A are accompanied by disparate transcriptional programs. MycT58A is associated with altered expression of genes underlying angiogenesis and inflammation, and MycS62A displays changes in expression of genes involved in maintaining genomic stability. Altogether, this study demonstrates that MycT58A and MycS62A induce unique pathophysiology in T-cell lymphomas, which may inform future research and T-ALL disease management strategies.

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies.

RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the E. coli HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for in vitro experiments. In this paper, we describe and compare methods for the economical (43.2 €/mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the Helicoverpa-specific gene Dicer2 and, as a non-endogenous control, the Green Fluorescent Protein gene (GFP). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates.

Hypoxia Regulates Endogenous Double-Stranded RNA Production via Reduced Mitochondrial DNA Transcription.

Hypoxia is a common phenomenon in solid tumours strongly linked to the hallmarks of cancer. Hypoxia promotes local immunosuppression and downregulates type I interferon (IFN) expression and signalling, which contribute to the success of many cancer therapies. Double-stranded RNA (dsRNA), transiently generated during mitochondrial transcription, endogenously activates the type I IFN pathway. We report the effects of hypoxia on the generation of mitochondrial dsRNA (mtdsRNA) in breast cancer. We found a significant decrease in dsRNA production in different cell lines under hypoxia. This effect was HIF1α/2α-independent. mtdsRNA was responsible for induction of type I IFN and significantly decreased after hypoxia. Mitochondrially encoded gene expression was downregulated and mtdsRNA bound by the dsRNA-specific J2 antibody was decreased during hypoxia. These findings reveal a new mechanism of hypoxia-induced immunosuppression that could be targeted by hypoxia-activated therapies.

Advances in the Development of Microbial Double-Stranded RNA Production Systems for Application of RNA Interference in Agricultural Pest Control.

RNA interference (RNAi) is a valuable and revolutionary technology that has been widely applied in medicine and agriculture. The application of RNAi in various industries requires large amounts of low-cost double-stranded RNA (dsRNA). Chemical synthesis can only produce short dsRNAs; long dsRNAs need to be synthesized biologically. Several microbial chassis cells, such as Escherichia coli, Saccharomyces cerevisiae, and Bacillus species, have been used for dsRNA synthesis. However, the titer, rate of production, and yield of dsRNA obtained by these microorganism-based strategies is still low. In this review, we summarize advances in microbial dsRNA production, and analyze the merits and faults of different microbial dsRNA production systems. This review provides a guide for dsRNA production system selection. Future development of efficient microbial dsRNA production systems is also discussed.

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector.

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.

Global Analysis of RNA-Dependent RNA Polymerase-Dependent Small RNAs Reveals New Substrates and Functions for These Proteins and SGS3 in Arabidopsis.

RNA silencing pathways control eukaryotic gene expression transcriptionally or posttranscriptionally in a sequence-specific manner. In RNA silencing, the production of double-stranded RNA (dsRNA) gives rise to various classes of 20–24 nucleotide (nt) small RNAs (smRNAs). In Arabidopsis thaliana, smRNAs are often derived from long dsRNA molecules synthesized by one of the six genomically encoded RNA-dependent RNA Polymerase (RDR) proteins. However, the full complement of the RDR-dependent smRNAs and functions that these proteins and their RNA-binding cofactors play in plant RNA silencing has not been fully uncovered. To address this gap, we performed a global genomic analysis of all six RDRs and two of their cofactors to find new substrates for RDRs and targets of the resulting RDR-derived siRNAs to uncover new functions for these proteins in plants. Based on these analyses, we identified substrates for the three RDRγ clade proteins (RDR3–5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, RDR2, and RDR6) as well as the RDR2 cofactor RNA-directed DNA methylation 12 (RDM12) and the RDR6 cofactor suppressor of gene silencing 3 (SGS3). These findings revealed that the target substrates of SGS3 are not limited to those solely utilized by RDR6, but that this protein seems to be a more general cofactor for the RDR family of proteins. Additionally, we found that RDR6 and SGS3 are involved in the production of smRNAs that target transcripts related to abiotic stresses, including water deprivation, salt stress, and ABA response, and as expected the levels of these mRNAs are increased in rdr6 and sgs3 mutant plants. Correspondingly, plants that lack these proteins (rdr6 and sgs3 mutants) are hypersensitive to ABA treatment, tolerant to high levels of PEG8000, and have a higher survival rate under salt treatment in comparison to wild-type plants. In total, our analyses have provided an extremely data-rich resource for uncovering new functions of RDR-dependent RNA silencing in plants, while also revealing a previously unexplored link between the RDR6/SGS3-dependent pathway and plant abiotic stress responses.

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli

ABSTRACT RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

C Protein is Essential for Canine Distemper Virus Virulence and Pathogenicity in Ferrets.

Paramyxoviruses, including members of the genus Morbillivirus, express accessory proteins with ancillary functions during viral replication. One of these, the C protein, is expressed from an alternate open reading frame (ORF) located in the P gene. The measles virus (MeV) C protein has been implicated in modulation of interferon signaling, but has more recently been shown to play a vital role in regulation of viral transcription and replication, preventing the excessive production of double-stranded RNA. Failure to do so, as seen with C-deficient MeV, leads to early activation of innate immune responses resulting in restriction of viral replication and attenuation in the host. One puzzling aspect of morbillivirus C protein biology has been the finding that a C-deficient canine distemper virus (CDV) generated with a similar mutagenesis strategy displayed no attenuation in ferrets, an animal model commonly used to evaluate CDV pathogenesis. To resolve how virus lacking this protein could maintain virulence, we re-visited the CDV C protein and found that truncated C proteins are expressed from the CDV gene using alternative downstream start codons even when the first start codon was disrupted. We introduced an additional point mutation abrogating expression of these truncated C proteins. A new CDV with this mutation was attenuated in vitro and led to increased activation of protein kinase R. It was also strongly attenuated in ferrets, inducing only mild disease in infected animals, thus replicating the phenotype of C-deficient MeV. Our results demonstrate the crucial role of morbillivirus C proteins in pathogenesis.IMPORTANCE The measles (MeV) and canine distemper viruses (CDV) express accessory proteins that regulate the host immune response and enhance replication. The MeV C protein is critical in preventing the generation of excess immunostimulatory double-stranded RNA. C protein-deficient MeV is strongly attenuated compared to wild-type virus, whereas CDV with a similarly disrupted C open reading frame is fully pathogenic. Here we show that CDV can compensate the disrupting mutations by expression of truncated, but apparently functional C proteins from several alternative start codons. We generated a new recombinant CDV that does not express these truncated C protein. This virus was attenuated both in cell culture and in ferrets, and finally resolves the paradox of the MeV and CDV C proteins, showing that both in fact have similar functions important for viral pathogenesis.

M6 A modification: A mechanism for protecting hematopoietic development in mammals.

N6 -methyladenosine (m6 A) is one of the most common internal modifications in messenger RNA, which is necessary for cell physiological activities. A recent study shows that during mammalian hematopoietic development, loss of m6 A modification leads to the aberrant production of double-stranded RNA, which results in the abnormal activation of innate immune response, and ultimately leads to hematopoietic failure. Accordingly, m6 A modification provide us an attractive direction for us to understand mammalian hematopoietic development and innate immune response.

Epigenetic Switch-Induced Viral Mimicry Evasion in Chemotherapy-Resistant Breast Cancer.

Tumor progression upon treatment arises from preexisting resistant cancer cells and/or adaptation of persister cancer cells committing to an expansion phase. Here, we show that evasion from viral mimicry response allows the growth of taxane-resistant triple-negative breast cancer (TNBC). This is enabled by an epigenetic state adapted to taxane-induced metabolic stress, where DNA hypomethylation over loci enriched in transposable elements (TE) is compensated by large chromatin domains of H3K27me3 to warrant TE repression. This epigenetic state creates a vulnerability to epigenetic therapy against EZH2, the H3K27me3 methyltransferase, which alleviates TE repression in taxane-resistant TNBC, leading to double-stranded RNA production and growth inhibition through viral mimicry response. Collectively, our results illustrate how epigenetic states over TEs promote cancer progression under treatment and can inform about vulnerabilities to epigenetic therapy. SIGNIFICANCE: Drug-resistant cancer cells represent a major barrier to remission for patients with cancer. Here we show that drug-induced metabolic perturbation and epigenetic states enable evasion from the viral mimicry response induced by chemotherapy in TNBC. These epigenetic states define a vulnerability to epigenetic therapy using EZH2 inhibitors in taxane-resistant TNBC.See related commentary by Janin and Esteller, p. 1258.This article is highlighted in the In This Issue feature, p. 1241.

Articles of Significant Interest in This Issue

Virus infection activates RNase L, an interferon (IFN)-regulated endoribonuclease, to cleave viral and cellular RNAs. The RNase L-cleaved, double-stranded RNA products induce formation of antiviral stress granules (avSGs) and recruit antiviral proteins RIG-I, PKR, OAS, and RNase L to avSG to amplify IFN production. Manivannan et al. (e00205-20) identified a novel role of avSGs induced by RNase L as an antiviral signaling hub to coordinate RNA ligands with cognate receptors to mount an effective host response during viral infection. These findings shed light on the importance of asSGs for host immunity.

Double-stranded RNAs targeting HvRPS18 and HvRPL13 reveal potential targets for pest management of the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.

RNA interference (RNAi) is a potential tool for plant protection against insect pests. The great challenge for effective pest control using RNAi in the field is the development of efficient and reliable methods for the production and delivery of double-stranded RNA (dsRNA). In the present study, we investigated the potential of feeding in vitro synthesized or bacterially expressed dsRNA to populations of the 28-spotted ladybeetle Henosepilachna vigintioctopunctata as a method of biological pest control. Ingestion of in vitro synthesized dsHvRPS18 or dsHvRPL13 led to significant down-regulation of the ribosomal protein-encoding genes HvRPS18 and HvRPL13, respectively, and significantly decreased the survival of H. vigintioctopunctata. Such silencing of HvRPS18 or HvRPL13 expression appeared to be partially dose-dependent and also inhibited the growth of H. vigintioctopunctata and significantly suppressed the expression of digestive enzyme-related genes. Finally, ingestion of bacterially expressed dsHvRPS18 or dsHvRPL13 induced significant mortality in the first and third instars, and in adults. The effectiveness of RNAi-based gene silencing in H. vigintioctopunctata provides a powerful reverse genetic tool for the functional annotation of its genes. This study demonstrates that HvRPS18 and HvRPL13 represent candidate genes for RNAi-based biological control of H. vigintioctopunctata. © 2020 Society of Chemical Industry.