dsRNA Species Research Articles

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

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.

Length-Dependent Modulation of B Cell Activating Factor Transcripts in Chicken Macrophage by Viral Double-Stranded RNA.

Viral double-stranded RNA (dsRNA) interacts with Retinoic-acid-inducible-gene-1 (RIG-1)-like receptors (RLRs) to induce type 1 interferons. Melanoma-derived-antigen-5 (MDA-5), an RLR, but not RIG-1, is found in chickens. Ducks express both RIG-1 and MDA-5, a possible cause of differences in susceptibility to influenza virus infection between chickens and ducks. Using the HD11 chicken macrophage cell line and an RT2 Profiler PCR-array system, we showed that high-molecular-weight poly(I:C), HMW-poly(I:C), upregulates CCL4, interferon-gamma, interleukin-1, and interleukin-6 mRNA transcripts. HMW-poly(I:C), an in vitro surrogate of long dsRNA species, also induces the upregulation of IL-12B and B cell activating factor (BAFF). Conversely, low-molecular-weight poly(I:C), LMW-poly(I:C) did not induce a distinct cytokine expression pattern. Nonetheless, co-transfection of LMW and HMW-poly(I:C) significantly reduced the upregulation of IL12B and BAFF by HMW-poly(I:C). These findings support previous studies that found no expression of RIG-1, a receptor for short dsRNA species, in chicken cells. Surprisingly, however, our data suggested that in the absence of RIG-1 in chicken macrophages, short dsRNA species may inhibit macrophage-mediated B cell development and survival by modulating the expression of BAFF without significantly reducing type 1 interferon response.

Abstract B002: A CRISPR/Cas9 screening strategy for identifying modifiers of the viral mimicry response

Abstract Viral mimicry is the induction of a cellular antiviral response triggered by endogenous nucleic acids rather than exogenous viral infection. Such nucleic acids are sensed by cytosolic pattern recognition receptors and interpreted by the cell as an infection, leading to activation of an antiviral signaling cascade that results in interferon (IFN) signaling and subsequent upregulation of interferon-stimulated genes (ISGs). While downstream effects of ISGs are diverse and vary from direct induction of apoptosis to adaptive immune system engagement, the end result of viral mimicry is the culling of the ‘infected’ cancer cell. The viral mimicry response was first described as a mechanism of the FDA-approved DNA demethylating agents azacytidine (AZA) and decitabine (DAC). By removing transcriptionally repressive DNA methylation, these compounds allow normally-silenced repetitive elements of the genome to be expressed as double-stranded RNA (dsRNA) species that trigger viral mimicry. Since then, a body of literature has emerged implicating viral mimicry as a common mechanism underlying many different therapies and drug targets, including histone modifiers (e.g., LSD1, EZH2) as well as non-epigenetic targets such as cell cycle regulators (e.g., CDK4/6). The diversity of viral mimicry-inducing targets highlights the need for a systematic and discovery-based method to identify novel drug targets. Here, we accomplish this through the development of a viral mimicry cell reporter system composed of a green fluorescent protein (GFP) reporter coupled to the interferon-stimulated response element (ISRE) for CRISPR/Cas9-based pooled library screening. Using ISRE stimulation and subsequent GFP expression as a readout for viral mimicry activation, flow cytometry-based methods can be used to quantify the population of cells undergoing viral mimicry. Exogenous treatments of type I IFN or the synthetic dsRNA analogue polyinosinic-polycytidylic acid in the absence or presence of the JAK/STAT inhibitor ruxolitinib were used in conjunction with flow cytometry to validate the robustness and specificity of the reporter. To date, two preliminary screens in colorectal and lung cancer cell lines (LIM1215 and A549, respectively) have been performed using this system with a pooled single guide RNA library targeting epigenetic modifiers. These proof-of-principle experiments revealed two candidate hits – the histone methyltransferase SETDB1 and the lysine demethylase KDM5C. Follow-up validation experiments confirm GFP expression and viral mimicry activation in cells with single-gene knockouts for SETDB1 and KDM5C and demonstrate enhanced activation in combination with decitabine, as well as provide rationale for further study of these hits as therapeutic targets. Our work thus far demonstrates the feasibility of our approach and indicates the utility of our system as a novel tool for studying viral mimicry biology. Citation Format: Raymond Chen, Ilias Ettayebi, Daniel D. De Carvalho. A CRISPR/Cas9 screening strategy for identifying modifiers of the viral mimicry response. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B002.

A novel sustainable platform for scaled manufacturing of double-stranded RNA biopesticides

RNA interference (RNAi) represents one of the most conserved pathways evolved by eukaryotic cells for regulating gene expression. RNAi utilises non-translatable double-stranded RNA (dsRNA) molecules to sequester or degrade mRNA molecules gene. In RNAi, specifically designed exogenous dsRNA delivered to the cell can silence a target gene, a phenomenon that has been exploited in many functional studies and explored in biopesticide applications. The search for safe and sustainable crop pest management options drives the need to offset the effect of inorganic pesticides on biodiversity. The prospect of replacing inorganic pesticides with dsRNA crop spray is gaining popularity, enhanced by its high-target specificity and low environmental impact. However, for dsRNA to reach the pesticide market, it must be produced cost-effectively and sustainably. In this paper, we develop a high-yield expression media that generates up to 15-fold dsRNA yield compared to existing expression media utilising 1 mM IPTG. We also optimise a low-cost purification method that generates high-quality and purified dsRNA. The developed method circumvents the need for hazardous chemical reagents often found in commercial kits or commercial nucleases to eliminate contaminating DNA or single-stranded RNA (ssRNA) species. We also demonstrate that the production platform is scalable, generating 6.29 mg dsRNA from 259 mg wet E. coli cell pellet. The results also provide structural insights into the heterogeneous dsRNA species within the microbial-derived dsRNA pool. Finally, we also show that the purified ‘naked’ dsRNA, without prior formulation, can induce insect toxicity under field conditions. This study provides a novel, complete, low-cost process dsRNA platform with potential for application in industrial dsRNA production.Graphical

RNA Granules in the Mitochondria and Their Organization under Mitochondrial Stresses.

The human mitochondrial genome (mtDNA) regulates its transcription products in specialised and distinct ways as compared to nuclear transcription. Thanks to its mtDNA mitochondria possess their own set of tRNAs, rRNAs and mRNAs that encode a subset of the protein subunits of the electron transport chain complexes. The RNA regulation within mitochondria is organised within specialised, membraneless, compartments of RNA-protein complexes, called the Mitochondrial RNA Granules (MRGs). MRGs were first identified to contain nascent mRNA, complexed with many proteins involved in RNA processing and maturation and ribosome assembly. Most recently, double-stranded RNA (dsRNA) species, a hybrid of the two complementary mRNA strands, were found to form granules in the matrix of mitochondria. These RNA granules are therefore components of the mitochondrial post-transcriptional pathway and as such play an essential role in mitochondrial gene expression. Mitochondrial dysfunctions in the form of, for example, RNA processing or RNA quality control defects, or inhibition of mitochondrial fission, can cause the loss or the aberrant accumulation of these RNA granules. These findings underline the important link between mitochondrial maintenance and the efficient expression of its genome.

Efficient production of long double-stranded RNAs applicable to agricultural pest control by Corynebacterium glutamicum equipped with coliphage T7-expression system

RNA-based pesticides exert their function by suppressing the expression of an essential gene in the target pest through RNA interference caused by double-stranded RNA (dsRNA). Here, we selected target genes for growth suppression of the solanaceous crop pests ladybird beetle (Henosepilachna vigintioctopunctata) and Colorado potato beetle (Leptinotarsa decemlineata)-the death-associated inhibitor of apoptosis protein 1 gene (diap1), and an orthologous gene of the COPI coatomer protein complex (copI), respectively. We constructed a cost-competitive overproduction system for dsRNA using Corynebacterium glutamicum as a host bacterium. The dsRNA expression unit was equipped with two sets of promoters and terminators derived from coliphage T7, and the convergent expression system was designed to be selectively transcribed by T7 RNA polymerase. This expression system efficiently overproduced both target dsRNAs. On culture in a jar fermentor, the yield of diap1-targeting dsRNA (approximately 360 bp) was > 1 g per liter of culture. Long-chain diap1-targeting dsRNAs (up to around 1 kbp) could be produced without a substantial loss of efficiency. dsRNA accumulated in C. glutamicum significantly suppressed larval growth of H. vigintioctopunctata. The dsRNA expression technology developed here is expected to substantially reduce dsRNA production costs. Our method can be applied for a wide range of industrial uses, including agricultural pest control.Key points• Overexpression of dsRNA was achieved in C. glutamicum using a coliphage T7 system.• The best strain produced > 1 g/L of the target dsRNA species, for use as an insecticide.• The developed system efficiently produced long dsRNA species, up to ~ 1 kbp.

Biology and chemistry of an Umbravirus like 2989 bp single stranded RNA as a possible causal agent for Opuntia stunting disease (engrosamiento de cladodios) - A Review

Perhaps the most economically important disease of Opuntia ficus indica fruit cacti in Mexicois the “engrosamiento de cladodios” or macho disease. The symptoms of this disease, whichhas been suggested to be caused by a phytoplasma, are severe stunting of cladodes, flowersand fruits. In the mid-1980s this disease appeared in commercial cactus fruit orchards ofD’Arrigo Bros near Gonzalez, California. It was performed more than 30 PCR-based tests forviruses as well as various extraction methods and polymerase chain reaction (PCR) tests forphytoplasmas but were unable to find any of the known viruses or mycoplasmas in thestrongly symptomatic cactus with this disease. As almost all plant viruses go through areplication phase involving double stranded RNA (dsRNA), a dsRNA extraction wasperformed and a dsRNA species of about 600 bp identified. Then, reverse-transcribed thedsRNA, amplified the resultant cDNA by PCR, and cloned and sequenced the 600 bpfragment that were identified in symptomatic tissue. When this sequence was compared totranslated DNA in the National Center for Biotechnology Information (NCBI) nucleotide database (BLAST analysis) it was most similar to the Tobacco bushy top virus (E score of 2e-39),which is a single stranded RNA virus with no DNA intermediate. Primers made from this 630bp fragment were used to extend this sequence to 2989 sequence. This sequence appears tobe a full-length sequence with three open reading frames (ORF) and is shorter than theclosest class of viruses, the Umbraviruses that can be spread by mechanical transmissionand by aphids. It was not possible to transmit the virus or symptoms mechanically. Over asix-year period using traditional PCR, this virus was found in hundreds of symptomatic cactibut not in non-symptomatic pads. RT-PCR has found low levels of this virus on nonsymptomaticcladodes (3.7 fg) on a symptomatic plant and much higher concentrations(1x102 to 1x105 fg) on symptomatic cladodes from the same plant. Black bean aphids (Aphis fabae), that are the vector for a closely related Umbravirus known as groundnut rosetta virus,have been routinely found on the unopened flowers of cactus. This Umbravirus was found inaphids feeding on symptomatic cladodes. As Umbraviruses cannot infect plants without acompanion Luteovirus, that provides the protein coat for the Umbravirus, degenerateLuteovirus primers were used and a probable incomplete Luteovirus-like 4797 bp sequencewas found on aphids feeding on symptomatic cactus. This Luteovirus was not found inOpuntia cladodes using PCR. A micro RNA assembly of six pooled symptomatic Opuntiasdid not find a contig that spanned the 4797 putative Luteovirus sequence, but somefragments as large as 44 bp were exact matches to the Luteovirus. As Umbraviruses occurthroughout the plant but Luteoviruses only occur in the phloem, lower Luteovirusconcentrations would be expected. Two successive one hour 60°C heat treatmentseliminated these symptoms on new growth that was also PCR negative. A 5839 bpPotexvirus was found in some of these cladodes but its presence was not correlated with anysymptoms. Similar symptomatic cacti in Italy, South Africa and Mexico should be examinedwith these primers and dsRNA to see if similar correlations between presence/absence of thisfragment and symptomatic plants can be obtained. It is suggested that this disease be knownas OSD (Opuntia Stunting Disease).

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation.

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrainthe development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Suppressing PARylation by 2',5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death.

High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis invitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.

Azacytidine Inhibits Megakaryopoiesis Via the Induction of Immunogenic RNA Species and Activation of Type-I Interferon Signaling

Management of hematologic disease- and therapy-related thrombocytopenia remains a serious clinical issue, especially in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The ribonucleoside and DNA-demethylating agent azacytidine (AZA), has proven useful for the treatment of patients with MDS and AML not eligible for stem cell transplantation. While low-dose AZA therapy induces clinical remissions in up to 50% of treated patients, it comes at the cost of aggravating pre-existing thrombocytopenia which is observed in a subset of patients; this can lead to increased bleeding and bleeding-associated mortality, and importantly, often requires dose modifications and delays of therapy. Thus, identification of strategies alleviating ineffective megakaryopoiesis will likely lead to increased therapeutic efficacy for patients with MDS/AML. Eltrombopag (EP), a second-generation small molecule thrombopoietin receptor (TPO-R) agonist was effective in raising platelet counts in patients with MDS as a single agent, as well as in combination with certain standard of care therapies. However, it failed to stimulate platelet production during the first four cycles of AZA treatment as uncovered by a recent phase III placebo-controlled clinical study (SUPPORT; NCT02158936). The goals of this study were to identify the cellular and molecular underpinnings of AZA-associated inhibition of megakaryopoiesis and to assess the ineffectiveness of EP in mitigating AZA treatment-associated thrombocytopenia. Our results demonstrate that at a clinically-equivalent and non-cytotoxic dose, AZA rapidly induces transient activation of interferon type I (IFN-I) signaling in various hematopoietic cell types, including stem and lineage-committed progenitor cells (HSPCs). We detected IFNα and IFNβ production and release using ELISA and intracellular flow cytometry on primary total mononuclear cell- and purified CD34-positive HSPC populations derived from cord blood, bone marrow from healthy volunteers or patients with MDS/AML. AZA-mediated activation of Type I IFNs in healthy control- and MDS/AML cells was preceded by an accumulation of double-stranded RNA (dsRNA) species and decreased total RNA cytosine methylation measured by immunocytochemistry and intracellular FACS analysis; this suggested that AZA triggered the accumulation of immunogenic RNA species which elicit an IFN-I response. In support, we found Toll like receptor 3 (TLR3) activation and phosphorylation of STAT1 in CD34+ HPSC, along with premature activation of Suppressor of Cytokine Signaling 1 (SOCS1), a well-known JAK/STAT-dependent signaling attenuator. This rapid AZA-induced viral mimicry response led to abrogation of thrombopoietin (TPO) or EP-stimulated TPO-R signaling and inhibition of ex vivo megakaryocyte progenitor proliferation quantified by colony formation in semi-solid medium. Importantly, inhibition of IFN-I signal activation using the JAK3 inhibitor decernotinib, the IFNα/β-blocking peptide, B18R, or RNA interference-mediated knock-down of SOCS1 counteracted the inhibitory effects of AZA on TPO-R stimulation and restored megakaryopoiesis. Given these observations, we pre-clinically tested a revised treatment protocol, in which primary cells were first exposed to AZA for four days followed by TPO-R stimulation using TPO or EP. This new treatment strategy alleviated AZA's inhibitory effects at the molecular and cellular levels, demonstrating that upon resolution of the AZA-mediated vial mimicry response, EP and TPO can effectively stimulate TPO-R signaling and megakaryopoiesis. Together, our data reveal a mechanistic basis of AZA-mediated inhibition of megakaryopoiesis in patients with MDS/AML. Additionally, we show that EP cannot overcome the megakaryopoiesis-inhibitory effects of acute IFN-I signaling activation upon AZA exposure. Findings of our study are consistent with and provide a molecular explanation for the observations made in the context of the SUPPORT study. In the future, it will be critical to better understand and potentially counteract the megakaryopoiesis-inhibitory effects by IFN-I pathway activation upon AZA therapy in patients with MDS/AML. Disclosures Okoye-Okafor: Novartis Pharmaceuticals: Research Funding. Pallaud:Novartis Pharmaceuticals: Employment. Marques Ramos:Novartis Pharmaceuticals: Employment. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding. Will:Novartis Pharmaceuticals: Research Funding.

Immune Sensing of Aeroallergen-Associated Double-Stranded RNA Triggers an IFN Response and Modulates Type 2 Lung Inflammation

The innate immune sensing of allergens or allergen-associated components regulate the development of type 2 inflammatory responses. However, the underlying molecular basis by which allergens or allergen-associated components are detected by innate immune receptors remains elusive. In this study, we report that the most common aeroallergen, house dust mite (HDM), harbors a dsRNA species (HDM-dsRNA) that can activate TLR3-mediated IFN responses and counteract the development of an uncontrolled type 2 immune response. We demonstrate that the mouse strains defective in the dsRNA-sensing pathways show aggravated type 2 inflammation defined by severe eosinophilia, elevated level of type 2 cytokines, and mucus overproduction in a model of allergic lung inflammation. The inability to sense HDM-dsRNA resulted in significant increases in airway hyperreactivity. We further show that the administration of the purified HDM-dsRNA at a low dose is sufficient to induce an immune response to prevent the onset of a severe type 2 lung inflammation. Collectively, these results unveil a new role for the HDM-dsRNA/TLR3-signaling axis in the modulation of a type 2 lung inflammation in mice.

The herpes simplex virus host shutoff (vhs) RNase limits accumulation of double stranded RNA in infected cells: Evidence for accelerated decay of duplex RNA.

The herpes simplex virus virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs and blunts host innate antiviral responses. Previous work demonstrated that cells infected with vhs mutants display enhanced activation of the host double-stranded RNA (dsRNA)-activated protein kinase R (PKR), implying that vhs limits dsRNA accumulation in infected cells. Confirming this hypothesis, we show that partially complementary transcripts of the UL23/UL24 and UL30/31 regions of the viral genome increase in abundance when vhs is inactivated, giving rise to greatly increased levels of intracellular dsRNA formed by annealing of the overlapping portions of these RNAs. Thus, vhs limits accumulation of dsRNA at least in part by reducing the levels of complementary viral transcripts. We then asked if vhs also destabilizes dsRNA after its initial formation. Here, we used a reporter system employing two mCherry expression plasmids bearing complementary 3’ UTRs to produce defined dsRNA species in uninfected cells. The dsRNAs are unstable, but are markedly stabilized by co-expressing the HSV dsRNA-binding protein US11. Strikingly, vhs delivered by super-infecting HSV virions accelerates the decay of these pre-formed dsRNAs in both the presence and absence of US11, a novel and unanticipated activity of vhs. Vhs binds the host RNA helicase eIF4A, and we find that vhs-induced dsRNA decay is attenuated by the eIF4A inhibitor hippuristanol, providing evidence that eIF4A participates in the process. Our results show that a herpesvirus host shutoff RNase destabilizes dsRNA in addition to targeting partially complementary viral mRNAs, raising the possibility that the mRNA destabilizing proteins of other viral pathogens dampen the host response to dsRNA through similar mechanisms.

Function of HNRNPC in breast cancer cells by controlling the dsRNA‐induced interferon response

Elevated expression of RNA binding protein HNRNPC has been reported in cancer cells, while the essentialness and functions of HNRNPC in tumors were not clear. We showed that repression of HNRNPC in the breast cancer cells MCF7 and T47D inhibited cell proliferation and tumor growth. Our computational inference of the key pathways and extensive experimental investigations revealed that the cascade of interferon responses mediated by RIG‐I was responsible for such tumor‐inhibitory effect. Interestingly, repression of HNRNPC resulted in accumulation of endogenous double‐stranded RNA (dsRNA), the binding ligand of RIG‐I. These up‐regulated dsRNA species were highly enriched by Alu sequences and mostly originated from pre‐mRNA introns that harbor the known HNRNPC binding sites. Such source of dsRNA is different than the recently well‐characterized endogenous retroviruses that encode dsRNA. In summary, essentialness of HNRNPC in the breast cancer cells was attributed to its function in controlling the endogenous dsRNA and the down‐stream interferon response. This is a novel extension from the previous understandings about HNRNPC in binding with introns and regulating RNA splicing.

Detection of viral induced double-stranded RNA intermediates in archival paraffin blocks

Double strand RNA (dsRNA) species were previously thought to be a ‘junk DNA’ equivalent are related to endogenous retroviral elements (ERV) inserted into the human genome. Genomic integration sites and blocking of these processes have been well documented in HIV and Hepatitis C related diseases. Viral integration into human genome has been shown in a range of solid tumors, but mechanisms less understood. The detection of dsRNA in the research setting has been largely limited to cell line models and controlled transfections. To better characterize function significance of ERV and interactions at the cellular level, we outline efforts to detect and quantify such viral genomic elements with a focus on archival clinical samples in the commonly stored paraffin block. Tissue blocks issues with clinical and biochemical documented viral infection and associated viral cytopathic changes were selected from the Department of Pathology and Biorepository and standard 4 micron sections were prepared. Two (2) commercially available antibodies raised to dsRNA fragments (J2 and K1 antibodies) were directly compared by chromogenic and immunofluorescent methods. In addition, downstream biomarkers as identified by literature searches include RIG-1 and IRF 7 as moderate probable success and MDA-5 and STAT 1 as higher level success. Correlative analysis of downstream pathway markers with labeling results of dsRNA antibodies suffers from lack of sensitive measures of dsRNA fragments as opposed to the downstream cellular pathway markers. This lack of concordance likely reflects the short nature of dsRNAs in decade old paraffin tissue and/or differences in abundance. Future work will focus on developing more sensitive probes or IF probe signal amplification. Prediction of distant recurrence-free survival in resectable lung adenocarcinoma

Prediction of distant recurrence-free survival in resectable lung adenocarcinoma

Double strand RNA (dsRNA) species were previously thought to be a ‘junk DNA’ equivalent are related to endogenous retroviral elements (ERV) inserted into the human genome. Genomic integration sites and blocking of these processes have been well documented in HIV and Hepatitis C related diseases. Viral integration into human genome has been shown in a range of solid tumors, but mechanisms less understood. The detection of dsRNA in the research setting has been largely limited to cell line models and controlled transfections. To better characterize function significance of ERV and interactions at the cellular level, we outline efforts to detect and quantify such viral genomic elements with a focus on archival clinical samples in the commonly stored paraffin block. Tissue blocks issues with clinical and biochemical documented viral infection and associated viral cytopathic changes were selected from the Department of Pathology and Biorepository and standard 4 micron sections were prepared. Two (2) commercially available antibodies raised to dsRNA fragments (J2 and K1 antibodies) were directly compared by chromogenic and immunofluorescent methods. In addition, downstream biomarkers as identified by literature searches include RIG-1 and IRF 7 as moderate probable success and MDA-5 and STAT 1 as higher level success. Correlative analysis of downstream pathway markers with labeling results of dsRNA antibodies suffers from lack of sensitive measures of dsRNA fragments as opposed to the downstream cellular pathway markers. This lack of concordance likely reflects the short nature of dsRNAs in decade old paraffin tissue and/or differences in abundance. Future work will focus on developing more sensitive probes or IF probe signal amplification. Prediction of distant recurrence-free survival in resectable lung adenocarcinoma

Mungbean yellow mosaic Indian virus encoded AC2 protein suppresses RNA silencing by inhibiting Arabidopsis RDR6 and AGO1 activities

RNA silencing refers to a conserved RNA-directed gene regulatory mechanism in a wide range of eukaryotes. It plays an important role in many processes including growth, development, genome stability, and antiviral defense in the plants. Geminivirus encoded AC2 is identified as an RNA silencing suppressor protein, however, the mechanism of action has not been characterized. In this paper, we elucidate another mechanism of AC2-mediated suppression activity of Mungbean Yellow Mosaic India Virus (MYMIV). The AC2 protein, unlike many other suppressors, does not bind to siRNA or dsRNA species and its suppression activity is mediated through interaction with key components of the RNA silencing pathway, viz., RDR6 and AGO1. AC2 interaction inhibits the RDR6 activity, an essential component of siRNA and tasi-RNA biogenesis and AGO1, the major slicing factor of RISC. Thus the study identifies dual sites of MYMIV-AC2 interference and probably accounts for its strong RNA silencing suppression activity.

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well.

Nanostructured RNAs for RNA interference.

We synthesized three types of nanostructured RNAs that induce RNA interference (RNAi): branched RNAs, dumbbell-shaped RNA, and circular double-stranded RNAs. All three nanostructured RNAs were transformed into double-stranded RNA of approximately 20 base pairs when they were treated with nuclease enzymes such as Dicer. These dsRNA species induced gene silencing when they are were introduced into mammalian cells.

The Innate Immune Sensor LGP2 Activates Antiviral Signaling by Regulating MDA5-RNA Interaction and Filament Assembly

Cytoplasmic pattern recognition receptors detect non-self RNAs during virus infections and initiate antiviral signaling. One receptor, MDA5, possesses essential signaling domains, but weak RNA binding. A second receptor, LGP2, rapidly detects diverse dsRNA species, but lacks signaling domains. Accumulating evidence suggests LGP2 and MDA5 work together to detect viral RNA and generate a complete antiviral response, but the basis for their cooperation has been elusive. Experiments presented here address this gap in antiviral signaling, revealing that LGP2 assists MDA5-RNA interactions leading to enhanced MDA5-mediated antiviral signaling. LGP2 increases the initial rate of MDA5-RNA interaction and regulates MDA5 filament assembly, resulting in the formation of more numerous, shorter MDA5 filaments that are shown to generate equivalent or greater signaling activity invivo than the longer filaments containing only MDA5. These findings provide a mechanism for LGP2 coactivation of MDA5 and a biological context for MDA5-RNA filaments in antiviral responses.