Caenorhabditis elegans Dicer acts with the RIG-I-like helicase DRH-1 and RDE-4 to cleave dsRNA.

Invertebrates use the endoribonuclease Dicer to cleave viral dsRNA during antiviral defense, while vertebrates use RIG-I-like Receptors (RLRs), which bind viral dsRNA to trigger an interferon response. While some invertebrate Dicers act alone during antiviral defense, Caenorhabditis elegans Dicer acts in a complex with a dsRNA binding protein called RDE-4, and an RLR ortholog called DRH-1. We used biochemical and structural techniques to provide mechanistic insight into how these proteins function together. We found RDE-4 is important for ATP-independent and ATP-dependent cleavage reactions, while helicase domains of both DCR-1 and DRH-1 contribute to ATP-dependent cleavage. DRH-1 plays the dominant role in ATP hydrolysis, and like mammalian RLRs, has an N-terminal domain that functions in autoinhibition. A cryo-EM structure indicates DRH-1 interacts with DCR-1's helicase domain, suggesting this interaction relieves autoinhibition. Our study unravels the mechanistic basis of the collaboration between two helicases from typically distinct innate immune defense pathways.

The dsRNA isolated from Escherichia coli infected with the MS2 bacteriophage induces the production of interferons.

Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-ß. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-ß to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.

Initiation of a ZAKα-dependent ribotoxic stress response by the innate immunity endoribonuclease RNase L.

RNase L is an endoribonuclease of higher vertebrates that functions in antiviral innate immunity. Interferons induce oligoadenylate synthetase enzymes that sense double-stranded RNA of viral origin leading to the synthesis of 2',5'-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L remodels the host cell transcriptome. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A is directly introduced into cells. Here, we report that RNase L activation by 2-5A causes a ribotoxic stress response involving the MAP kinase kinase kinase (MAP3K) ZAKα, MAP2Ks, and the stress-activated protein kinases JNK and p38α. RNase L activation profoundly alters the transcriptome by widespread depletion of mRNAs associated with different cellular functions but also by JNK/p38α-stimulated induction of inflammatory genes. These results show that the 2-5A/RNase L system triggers a protein kinase cascade leading to proinflammatory signaling and apoptosis.

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing.

Cytosolic long dsRNA, among the most potent proinflammatory signals, is recognized by melanoma differentiation-associated protein 5 (MDA5). MDA5 binds dsRNA cooperatively forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-ß responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. Consecutive rounds of ATP hydrolysis amplify the filament footprint, displacing tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling identifies an internally bound MDA5-LGP2-RNA complex, with the LGP2 C-terminal tail forming the key contacts with MDA5. These contacts are specifically required for NTP-dependent internal RNA binding. We conclude that NTPase-dependent binding of LGP2 to internal dsRNA sites complements NTPase-independent binding to dsRNA ends, via distinct binding modes, to increase the number and signaling output of MDA5-dsRNA complexes.

Lethal and sublethal effects of orally delivered double-stranded RNA on the cabbage stem flea beetle, Psylliodes chrysocephala.

BACKGROUND: The cabbage stem flea beetle (Psylliodes chrysocephala) is one of the most important insect pests of oilseed rape (Brassica napus) in northern Europe. The emergence of insecticide-resistant populations and the ban on neonicotinoid seed treatments have made the management of this pest challenging and research is needed to develop alternative strategies such as RNA interference (RNAi). We investigated lethal and sublethal effects of orally delivered double-stranded (ds)RNAs targeting P. chrysocephala orthologs of Sec23 and vacuolar adenosine triphosphatase subunit G (VatpG), which are involved in endoplasmic reticulum-Golgi transport and organelle acidification, respectively. RESULTS: Feeding bioassays on P. chrysocephala adults showed that the highest concentration (200 ng/leaf disk) of dsSec23 caused mortalities of 76% and 56% in pre-aestivating and post-aestivating beetles, respectively, while the same concentration of dsVatpG led to mortality rates of ~34% in both stages. Moreover, sublethal effects, such as decreased feeding rates and attenuated locomotion were observed. Small RNA sequencing and gene expression measurements following the delivery of dsRNAs demonstrated the generation of ~21 nucleotide-long small interfering RNAs and a systemic RNAi response in P. chrysocephala. CONCLUSION: We demonstrate that P. chrysocephala is a promising candidate for developing RNAi-based pest management strategies. Further research is necessary to identify more effective target genes and to assess potential non-target effects. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Atypical activation of the RNA sensor MDA5 by hepatitis C virus.

Hepatitis C virus (HCV) is a significant human pathogen that can cause a number of serious diseases including chronic inflammation of the liver, cirrhosis, and hepatocellular carcinoma. A key enzyme in the HCV life cycle is the nonstructural protein 5B (NS5B), which functions as an RNA-dependent RNA polymerase (RdRp) responsible for replicating the viral RNA genome. In their recent study, Dansako and colleagues showed that HCV NS5B induces type I interferon via activation of the RNA receptor MDA5, an activity that was dependent on the RdRp enzymatic activity but independent of viral RNA replication. Their data further indicated that the NS5B enzymes of HCV and the related GB virus-B produce cellular double-stranded RNA (dsRNA) species with potential immunostimulatory activity. These findings unveil an unconventional mechanism of activation of MDA5-mediated host immunity by viral RdRp enzymes, which is expected to spur new research directions in viral immunology.

Structural Studies of Bacteriophage Φ6 and Its Transformations during Its Life Cycle.

From the first isolation of the cystovirus bacteriophage Φ6 from Pseudomonas syringae 50 years ago, we have progressed to a better understanding of the structure and transformations of many parts of the virion. The three-layered virion, encapsulating the tripartite double-stranded RNA (dsRNA) genome, breaches the cell envelope upon infection, generates its own transcripts, and coopts the bacterial machinery to produce its proteins. The generation of a new virion starts with a procapsid with a contracted shape, followed by the packaging of single-stranded RNA segments with concurrent expansion of the capsid, and finally replication to reconstitute the dsRNA genome. The outer two layers are then added, and the fully formed virion released by cell lysis. Most of the procapsid structure, composed of the proteins P1, P2, P4, and P7 is now known, as well as its transformations to the mature, packaged nucleocapsid. The outer two layers are less well-studied. One additional study investigated the binding of the host protein YajQ to the infecting nucleocapsid, where it enhances the transcription of the large RNA segment that codes for the capsid proteins. Finally, I relate the structural aspects of bacteriophage Φ6 to those of other dsRNA viruses, noting the similarities and differences.

Involvement of Protein Kinase R in Double-Stranded RNA-Induced Proteasomal Degradation of Hypoxia Inducible Factor-1α.

Hypoxia inducible factor-1α (HIF-1α) is a crucial therapeutic target in various diseases, including cancer and fibrosis. We previously demonstrated that transfection with double-stranded RNA (dsRNA), including polyI:C and the dsRNA genome of mammalian orthoreovirus, resulted in significant reduction in HIF-1α protein levels in cultured cells; however, it remained to be elucidated how dsRNA induced down-regulation of HIF-1α protein levels. In this study, we examined the mechanism of dsRNA-mediated down-regulation of HIF-1α protein levels. We found that among the various cellular factors involved in dsRNA-mediated innate immunity, knockdown and knockout of protein kinase R (PKR) significantly restored HIF-1α protein levels in dsRNA-transfected cells, indicating that PKR was involved in dsRNA-mediated down-regulation of HIF-1α. Proteasome inhibitors significantly restored the HIF-1α protein levels in dsRNA-transfected cells. Ubiquitination levels of HIF-1α were increased by transfection with dsRNA. These findings indicated that degradation of HIF-1α in a ubiquitin-proteasome pathway was promoted in a PKR-dependent manner following dsRNA transfection. Expression of not only HIF-1α but also several proteins, including CDK4 and HER2, was down-regulated following dsRNA transfection. These data provide important clues for elucidation of the mechanism of dsRNA-mediated cellular toxicity, as well as for therapeutic application of dsRNA.

The role of protein kinase R in placental inflammation, mtUPR and apoptosis.

INTRODUCTION: Placental inflammation is implicated in the pathophysiology of many pregnancy complications, including fetal growth restriction, preeclampsia, gestational diabetes, and choriocarcinoma. Mitochondrial dysfunction, one of the outcomes of placental inflammation, is characterized by loss of membrane potential, accumulation of oxygen radicals, mitochondrial protein folding defects, and disturbances in mitochondrial dynamics. Protein kinase R (PKR) is stimulated by double-stranded RNA and bacterial endotoxins in the presence of pathogens and is a critical immune response enzyme. PKR is also correlated with the cell death response during endoplasmic reticulum stress. In this study, we aim to investigate the effects of PKR activity stimulated by lipopolysaccharide (LPS), and double-stranded RNA analog (Poly I:C) on mitochondrial unfolded protein response (mtUPR), mitochondrial membrane potential, apoptosis, and oxidative stress in placental trophoblasts. METHODS: We applied LPS and Poly I:C to BeWo cells to induce PKR activation. In addition, cells were treated with 2-aminopurine (2-AP) to inhibit the kinase activity of PKR. Protein levels of ATP-dependent Clp protease proteolytic subunit (CLPP) and heat shock protein 60 (HSP60) were determined after treatments. Apoptotic markers were detected by real-time PCR and flow cytometry. PKR-induced reactive oxygen radicals (ROS) accumulation and mitochondrial membrane potential change were assessed by flow cytometry. RESULTS: It was determined that PKR activation-induced apoptosis in BeWo cells by reducing the levels of mtUPR proteins (CLPP and HSP60) and caused a decrease in mitochondrial membrane potential. PKR inhibition was sufficient for decreases in apoptotic markers and caused a reduction in the ratio of depolarized and ROS (+) cells. DISCUSSION: Our results showed that LPS and Poly I:C administration stimulated PKR in BeWo cells in vitro. Furthermore, PKR activation is correlated with the levels of proteins involved in mitochondrial homeostasis and apoptosis. Our findings will contribute to understanding the role of PKR activation in placental inflammation and related diseases.

Plastid dsRNA transgenes trigger phased small RNA-based gene silencing of nuclear-encoded genes.

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis.

Resveratrol Attenuates the Mitochondrial RNA-Mediated Cellular Response to Immunogenic Stress.

Human mitochondria contain a circular genome that encodes 13 subunits of the oxidative phosphorylation system. In addition to their role as powerhouses of the cells, mitochondria are also involved in innate immunity as the mitochondrial genome generates long double-stranded RNAs (dsRNAs) that can activate the dsRNA-sensing pattern recognition receptors. Recent evidence shows that these mitochondrial dsRNAs (mt-dsRNAs) are closely associated with the pathogenesis of human diseases that accompany inflammation and aberrant immune activation, such as Huntington's disease, osteoarthritis, and autoimmune Sjögren's syndrome. Yet, small chemicals that can protect cells from a mt-dsRNA-mediated immune response remain largely unexplored. Here, we investigate the potential of resveratrol (RES), a plant-derived polyphenol with antioxidant properties, on suppressing mt-dsRNA-mediated immune activation. We show that RES can revert the downstream response to immunogenic stressors that elevate mitochondrial RNA expressions, such as stimulation by exogenous dsRNAs or inhibition of ATP synthase. Through high-throughput sequencing, we find that RES can regulate mt-dsRNA expression, interferon response, and other cellular responses induced by these stressors. Notably, RES treatment fails to counter the effect of an endoplasmic reticulum stressor that does not affect the expression of mitochondrial RNAs. Overall, our study demonstrates the potential usage of RES to alleviate the mt-dsRNA-mediated immunogenic stress response.

RETINOBLASTOMA-RELATED interactions with key factors of the RNA-directed DNA methylation (RdDM) pathway and its influence on root development.

MAIN CONCLUSION: Our study presents evidence for a novel mechanism for RBR function in transcriptional gene silencing by interacting with key players of the RdDM pathway in Arabidopsis and several plant clades. Transposable elements and other repetitive elements are silenced by the RNA-directed DNA methylation pathway (RdDM). In RdDM, POLIV-derived transcripts are converted into double-stranded RNA (dsRNA) by the activity of RDR2 and subsequently processed into 24 nucleotide short interfering RNAs (24-nt siRNAs) by DCL3. 24-nt siRNAs serve as guides to direct AGO4-siRNA complexes to chromatin-bound POLV-derived transcripts generated from the template/target DNA. The interaction between POLV, AGO4, DMS3, DRD1, RDM1 and DRM2 promotes DRM2-mediated de novo DNA methylation. The Arabidopsis Retinoblastoma protein homolog (RBR) is a master regulator of the cell cycle, stem cell maintenance, and development. We in silico predicted and explored experimentally the protein-protein interactions (PPIs) between RBR and members of the RdDM pathway. We found that the largest subunits of POLIV and POLV (NRPD1 and NRPE1), the shared second largest subunit of POLIV and POLV (NRPD/E2), RDR1, RDR2, DCL3, DRM2, and SUVR2 contain canonical and non-canonical RBR binding motifs and several of them are conserved since algae and bryophytes. We validated experimentally PPIs between Arabidopsis RBR and several of the RdDM pathway proteins. Moreover, seedlings from loss-of-function mutants in RdDM and RBR show similar phenotypes in the root apical meristem. We show that RdDM and SUVR2 targets are up-regulated in the 35S:AmiGO-RBR background.

Engineered Polymer-siRNA Polyplexes Provide Effective Treatment of Lung Inflammation.

Uncontrolled inflammation is responsible for acute and chronic diseases in the lung. Regulating expression of pro-inflammatory genes in pulmonary tissue using small interfering RNA (siRNA) is a promising approach to combatting respiratory diseases. However, siRNA therapeutics are generally hindered at the cellular level by endosomal entrapment of delivered cargo and at the organismal level by inefficient localization in pulmonary tissue. Here we report efficient anti-inflammatory activity in vitro and in vivo using polyplexes of siRNA and an engineered cationic polymer (PONI-Guan). PONI-Guan/siRNA polyplexes efficiently deliver siRNA cargo to the cytosol for highly efficient gene knockdown. Significantly, these polyplexes exhibit inherent targeting to inflamed lung tissue following intravenous administration in vivo. This strategy achieved effective (>70%) knockdown of gene expression in vitro and efficient (>80%) silencing of TNF-α expression in lipopolysaccharide (LPS)-challenged mice using a low (0.28 mg/kg) siRNA dosage.

Sequence determinant of small RNA production by DICER.

RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs)1,2. However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3' overhang and a terminal loop3-11. Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis-acting element, termed the 'GYM motif' (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified 'ruler'-like counting mechanisms from the 5' and 3' ends of pre-miRNA3-6. Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics.

Structure of the human DICER-pre-miRNA complex in a dicing state.

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs-unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified 'GYM motif'3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5' end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5' terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5' pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

Strengthening of the barrier function in human telomerase reverse transcription (hTERT) immortalized corneal and conjunctival epithelium by double-stranded RNA.

To investigate the response to polyinosinic:polycytidylic acid [poly(I:C)], a double-stranded RNA Toll-like receptor 3 agonist that mimics viral infection, in the barrier function of two established human telomerase reverse transcriptase-immortalized cell lines, termed HCLE for the human corneal-limbal epithelial line and HCjE for the human conjunctival-epithelial line. In this study, HCLE and HCjE cells were used to evaluate the underlying mechanism of epithelial-cell barrier function regulation. Briefly, HCLE and HCjE cells were first cultured on 12-well Transwell® (Corning®) filter-plates, and reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical examinations were then performed to assess tight junction (TJ)-related protein expression and cellular distribution. Next, the barrier function of the cells was measured via transepithelial electrical resistance (TEER) and paracellular molecular flux. The cells were then stimulated with poly(I:C) and the TEER and TJ-related protein expressions were analyzed. Similar to that in in vivo epithelium, the expression of claudin (CLDN) subtypes CLDN-1, -4, and -7 was observed in the HCLE and HCjE cells, and the barrier function in the HCLE cells was tighter than that in the HCjE cells. Post stimulation with poly(I:C), TEER of the HCLE and HCjE cells increased in a dose- and time-dependent manner, the production of TJ-related protein mRNA and CLDN-4 protein were elevated, and the barrier function of the HCLE and HCjE cells increased, thus possibly indicating that the increased barrier function is a defense mechanism against viral infection.

Effect of TLR3/dsRNA complex inhibitor on Poly(I:C)-induced airway inflammation in Swiss albino mice.

Rhinovirus infection frequently causes COPD and asthma exacerbations. Impaired anti-viral signaling and reduced viral clearance have both been seen in sick bronchial epithelium, potentially increasing exacerbations. Polyinosinic:polycytidylic acid (Poly(I:C)), a Toll-like receptor-3 (TLR3) ligand, has been shown to cause a viral exacerbation of severe asthma by detecting double-stranded RNA (dsRNA). The purpose of this work was to determine the effect of a TLR3/dsRNA complex inhibitor-Calbiochem drug in the prevention of Poly(I:C)-induced airway inflammation following TLR3 activation and to uncover a potential pathway for the cure of asthma through TLR3 inhibition. Mice were sensitized with Poly(I:C) as an asthma model before being challenged by PBS and ovalbumin (OVA) chemicals. The mice were administered a TLR3/dsRNA complex inhibitor. Throughout the trial, the mice's body weight was measured after each dosage. Biochemical methods are used to analyze the protein as well as enzyme composition in airway tissues. BALF specimens are stained using Giemsa to identify inflammatory cells and lung histopathology to determine morphological abnormalities in lung tissues. By using the ELISA approach, cytokine levels such as TNF-α, IL-13, IL-6, IL-5, and IgE antibody expression in lung tissue and blood serum were assessed. TLR3/dsRNA complex inhibitor drug significantly lowered the number of cells in BALF and also on Giemsa staining slides. It also downregulated the level of TNF-α and IL-6 in contrast to OVA and Poly(I:C) administered in animals. A TLR3/dsRNA complex inhibitor decreased the fraction of oxidative stress markers (MDA, GSH, GPx, and CAT) in lung tissues while keeping the mice's body weight constant during the treatment period. By decreasing alveoli, bronchial narrowing, smooth muscle hypertrophy, and granulocyte levels, the TLR3/dsRNA complex blocker significantly reduced the histopathological damage caused by OVA and Poly(I:C) compounds. In an animal model utilizing ovalbumin, TLR3/dsRNA complex inhibitors similarly reduced the bronchial damage produced by Poly(I:C). A novel TLR3/dsRNA complex inhibitor is expected to be employed in clinical studies since it suppresses airway inflammation without inducing antiviral approach resistance.

Theilovirus 3C Protease Cleaves the C-Terminal Domain of the Innate Immune RNA Sensor, Melanoma Differentiation-Associated Gene 5, and Impairs Double-Stranded RNA-Mediated IFN Response.

Melanoma differentiation-associated gene 5 (MDA5), a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), has pivotal roles in innate immune responses against many positive-stranded RNA viruses, including picornavirus and coronavirus. Upon engagement with dsRNA derived from viral infection, MDA5 initiates coordinated signal transduction leading to type I IFN induction to restrict viral replication. In this study, we describe a targeted cleavage events of MDA5 by the 3C protease from Theilovirus. Upon ectopic expression of theilovirus 3C protease from Saffold virus or Theiler's murine encephalomyelitis virus but not encephalomyocarditis virus, fragments of cleaved MDA5 were observed in a dose-dependent manner. When enzymatically inactive Theilovirus 3C protease was expressed, MDA5 cleavage was completely abrogated. Mass spectrometric analysis identified two cleavage sites at the C terminus of MDA5, cleaving off one of the RNA-binding domains. The same cleavage pattern was observed during Theilovirus infection. The cleavage of MDA5 by Theilovirus protease impaired ATP hydrolysis, RNA binding, and filament assembly on RNA, resulting in dysfunction of MDA5 as an innate immune RNA sensor for IFN induction. Furthermore, the cleavage-resistant MDA5 mutant against the 3C protease showed an enhanced IFN response during Saffold virus infection, indicating that Theilovirus has a strategy to circumvent the antiviral immune response by cleaving MDA5 using 3C protease. In summary, these data suggest MDA5 cleavage by 3C protease as a novel immune evasive strategy of Theilovirus.

Simultaneous Production of a Virus-Like Particle Linked to dsRNA to Enhance dsRNA Delivery for Yellow Head Virus Inhibition.

A co-expressed Penaeus stylirostris densovirus (PstDNV) capsid and dsRNA specific to the yellow head virus (YHV) protease (CoEx cpPstDNV/dspro) has been shown to suppress YHV replication in the Pacific white-legged shrimp (Litopenaeus vannamei). However, maintaining two plasmids in a single bacterial cell is not desirable; therefore, a single plasmid harboring both the PstDNV capsid and the dsRNA-YHV-pro gene was constructed under the regulation of a single T7 promoter, designated pET28a-Linked cpPstDNV-dspro. Following induction, this novel construct expressed an approximately 37-kDa recombinant protein associated with a roughly 400-bp dsRNA (Linked cpPstDNV-dspro). Under a transmission electron microscope, the virus-like particles (VLP; Linked PstDNV VLPs-dspro) obtained were seen to be monodispersed, similar to the native PstDNV virion. A nuclease digestion assay indicated dsRNA molecules were both encapsulated and present outside the Linked PstDNV VLPs-dspro. In addition, the amount of dsRNA produced from this strategy was higher than that obtained with a co-expression strategy. In a YHV infection challenge, the Linked PstDNV VLPs-dspro was more effective in delaying and reducing mortality than other constructs tested. Lastly, the linked construct provides protection for the dsRNA cargo from nucleolytic enzymes present in the shrimp hemolymph. This is the first report of a VLP carrying virus-inhibiting dsRNA that could be produced without disassembly and reassembly to control virus infection in shrimp.

Selective extracellular secretion of small double-stranded RNA by Tetragenococcus halophilus.

Small double-stranded RNAs (dsRNAs) abundantly produced by lactic acid bacteria demonstrate immunomodulatory activity and antiviral protective immunity. However, the extracellular secretion of dsRNA from lactic acid bacteria and their compositional and functional differences compared to the intracellular dsRNA is unknown. In this study, we compared the intracellular and secreted extracellular dsRNA of the lactic acid bacteria, Tetragenococcus halophilus, commonly present in fermented foods, by growing in RNA-free and RNase-free media. We used RNA deep sequencing and in-silico analysis to annotate potential regulatory functions for the comparison. A time series sampling of T. halophilus culture demonstrated growth phase-dependent dynamics in extracellular dsRNA secretion with no major change in the intracellular dsRNA profile. The RNA deep sequencing resulted in thousands of diverse dsRNA fragments with 14-21 nucleotides in size from T. halophilus culture. Over 70% of the secreted extracellular dsRNAs were unique in their sequences compared to the intracellular dsRNAs. Furthermore, the extracellular dsRNA abundantly contains sequences that are not T. halophilus genome encoded, not detected intracellularly and showed higher hits on human transcriptome during in-silico analysis, which suggests the presence of extrachromosomal mobile regulatory elements. Further analysis showed significant enrichment of dsRNA target genes of human transcriptome on cancer pathways and transcription process, indicating the extracellular dsRNA of T. halophilus is different not only at the sequence level but also in function. Studying the bacterial extracellular dsRNA is a promising area of future research, particularly for developing postbiotic fermented functional foods and understanding the impact of commensal gut bacteria on human health.