dsRNA Binding Research Articles

PH-Dependent Assembly and Stability of Toll-Like Receptor 3/dsRNA Signaling Complex: Insights from Constant pH Molecular Dynamics and Metadynamics Simulations.

The pH-dependent assembly of Toll-like receptors (TLRs), which triggers a threshold-like response, is a key principle in immune signaling. While crystallography has revealed the intricate structure of these assembly complexes, the mechanisms underlying their pH dependency remain unclear. Herein, constant pH simulations and metadynamics are employed to investigate the pH-dependent assembly and stability of the TLR3/dsRNA signaling complex. The findings demonstrate that system pH regulates complex assembly and stability by modulating the protonation and charge states of histidines. Histidines in TLR3 act as pH-dependent, positively charged binding sites that capture negatively charged dsRNA. Additionally, these histidines form a [H682⁺]-[E626⁻] dipole, facilitating the assembly of two TLR3 molecules into an antisymmetric dimer through dipole-dipole interactions. Surprisingly, TLR3 can shift the pKa values of key histidines from their model pKa of 6.5, increasing protonation likelihood and enhancing ligand binding. Notably, the aromatic residue Phe84, located within the dsRNA binding site [His39⁺-His60⁺-Phe84-His108⁺], alters the pKa of His60 through cation-π interactions with its protonated state. This study offers new insights into the molecular mechanisms underlying pH-dependent immune signaling via higher-order assemblies and suggests potential applications for histidine in self-assembling biomaterials.

Opening and closing of a cryptic pocket in VP35 toggles it between two different RNA-binding modes.

Cryptic pockets are of growing interest as potential drug targets, particularly to control protein-nucleic acid interactions that often occur via flat surfaces. However, it remains unclear whether cryptic pockets contribute to protein function or if they are merely happenstantial features that can easily be evolved away to achieve drug resistance. Here, we explore whether a cryptic pocket in the Interferon Inhibitory Domain (IID) of viral protein 35 (VP35) of Zaire ebolavirus aids its ability to bind double-stranded RNA (dsRNA). We use simulations and experiments to study the relationship between cryptic pocket opening and dsRNA binding of the IIDs of two other filoviruses, Reston and Marburg. These homologs have nearly identical structures but block different interferon pathways due to different affinities for blunt ends and backbone of the dsRNA. Simulations and thiol-labeling experiments demonstrate that the homologs have varying probabilities of pocket opening. Subsequent dsRNA-binding assays suggest that closed conformations preferentially bind dsRNA blunt ends while open conformations prefer binding the backbone. Point mutations that modulate pocket opening proteins further confirm this preference. These results demonstrate the open cryptic pocket has a function, suggesting cryptic pockets are under selective pressure and may be difficult to evolve away to achieve drug resistance.

Activation of PKR by a short-hairpin RNA

Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54’s potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation.

The making of a nucleic acid sensor at the dawn of jawed vertebrate evolution.

Self and nonself discrimination is fundamental to immunity. However, it remains largely enigmatic how the mechanisms of distinguishing nonself from self originated. As an intracellular nucleic acid sensor, protein kinase R (PKR) recognizes double-stranded RNA (dsRNA) and represents a crucial component of antiviral innate immunity. Here, we combine phylogenomic and functional analyses to show that PKR proteins probably originated from a preexisting kinase protein through acquiring dsRNA binding domains at least before the last common ancestor of jawed vertebrates during or before the Silurian period. The function of PKR appears to be conserved across jawed vertebrates. Moreover, we repurpose a protein closely related to PKR proteins into a putative dsRNA sensor, recapturing the making of PKR. Our study illustrates how a nucleic acid sensor might have originated via molecular tinkering with preexisting proteins and provides insights into the origins of innate immunity.

Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding.

Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.

An ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA inhibits PKR activation

Adar null mutant mouse embryos die with aberrant double-stranded RNA (dsRNA)-driven interferon induction, and Adar Mavs double mutants, in which interferon induction is prevented, die soon after birth. Protein kinase R (Pkr) is aberrantly activated in Adar Mavs mouse pup intestines before death, intestinal crypt cells die, and intestinal villi are lost. Adar Mavs Eifak2 (Pkr) triple mutant mice rescue all defects and have long-term survival. Adenosine deaminase acting on RNA 1 (ADAR1) and PKR co-immunoprecipitate from cells, suggesting PKR inhibition by direct interaction. AlphaFold studies on an inhibitory PKR dsRNA binding domain (dsRBD)-kinase domain interaction before dsRNA binding and on an inhibitory ADAR1 dsRBD3-PKR kinase domain interaction on dsRNA provide a testable model of the inhibition. Wild-type or editing-inactive human ADAR1 expressed in A549 cells inhibits activation of endogenous PKR. ADAR1 dsRNA binding is required for, but is not sufficient for, PKR inhibition. Mutating the ADAR1 dsRBD3-PKR contact prevents co-immunoprecipitation, ADAR1 inhibition of PKR activity, and co-localization of ADAR1 and PKR in cells.

Improving grapevine virus diagnostics: Comparative analysis of three dsRNA enrichment methods for high-throughput sequencing

The extraction of double stranded (ds) RNA is a common enrichment method for the study, characterization, and detection of RNA viruses. In addition to RNA viruses, viroids, and some DNA viruses, can also be detected from dsRNA enriched extracts which makes it an attractive method for detecting a wide range of viruses when coupled with HTS. Several dsRNA enrichment strategies have been developed. The oldest utilizes the selective binding properties of dsRNA to cellulose. More recent methods are based on the application of anti-dsRNA antibodies and viral proteins with a specific affinity for dsRNA. All three methods have been used together with HTS for plant virus detection and study. To our knowledge, this is the first comparative study of three alternative dsRNA enrichment methods for virus and viroid detection through HTS using virus-infected, and healthy grapevine test plants. Extracts were performed in triplicate using methods based on, the anti-dsRNA antibody mAb rJ2 (Millipore Sigma Canada Ltd, Oakville, ON, Canada), the B2 dsRNA binding protein, and ReliaPrep™ Resin (Promega Corporation, Madison, WI, USA). The results show that the workflows for all three methods are effectively comparable, apart from purification steps related to antibody and binding protein construct. Both the cellulose resin and dsRNA binding protein construct methods provide highly enriched dsRNA extracts suitable for HTS with the B2 method providing a 36× and the ReliaPrep™ Resin a 163× increase in dsRNA enrichment compared to the mAb rJ2 antibody. The overall consistency and cost effectiveness of the ReliaPrep™ cellulose resin-based method and the potentially simpler adaptation to robotics made it the method of choice for future transfer to a semi-automated workflow.

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.

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.

CgADAR1 involved in regulating the synthesis of interferon-like protein in Crassostrea gigas

Adenosine deaminases acting on RNA 1 (ADAR1) is a dsRNA adenosine (A)-to-inosine (I) editing enzyme that regulates the innate immune response against virus invasion. In the present study, a novel CgADAR1 was identified from the oyster Crassostrea gigas. The open reading frame (ORF) of CgADAR1 was of 3444 bp encoding a peptide of 1147 amino acid residues with two Zα domains, one dsRNA binding motif (DSRM) and one RNA adenosine deaminase domain (ADEAMc). The mRNA transcripts of CgADAR1 were detected in all the examined tissues, with higher expression levels in mantle and gill, which were 7.11-fold and 4.90-fold (p < 0.05) of that in labial palp, respectively. The mRNA transcripts of CgADAR1 in haemocytes were significantly induced at 24 h and 36 h after Poly (A: U) stimulation, which were 6.03-fold (p < 0.01) and 1.37-fold (p < 0.001) of that in control group, respectively. At 48 h after Poly (A:U) stimulation, the mRNA expression of CgRIG-Ⅰ, CgIRF8 and CgIFNLP significantly increased, which were 4.36-fold (p < 0.001), 1.82-fold (p < 0.05) and 1.92-fold (p < 0.05) of that in control group. After CgADAR1 expression was inhibited by RNA interference (RNAi), the mRNA expression levels of CgMDA5, CgRIG-Ⅰ, CgTBK1, CgIRF8 and CgIFNLP were significantly increased, which were 11.88-fold, 11.51-fold, 2.22-fold, 2.85-fold and 2.52-fold of that in control group (p < 0.001), and the phosphorylation level of CgTBK1 was also significantly increased. These results suggested that CgADAR1 played a regulation role in the early stages of viral infection by inhibiting the synthesis of interferon-like protein.

Chemical shift assignment of dsRBD1 and dsRBD2 of Arabidopsis thaliana DRB3, an essential protein involved in RNAi-mediated antiviral defense.

As sessile organisms, plants need to counteract different biotic and abiotic stresses to survive. RNA interference provides natural immunity against various plant pathogens, especially against viral infections via inhibition of viral genome replication or translation. In plants, DRB3, a multi-domain protein containing two N-terminal dsRNA binding domains (dsRBD), plays a vital role in RNA-directed DNA methylation of the geminiviral genome. Additionally, DRB3 arrests the replication of the viral genome in the viral replication complex of RNA viruses through a mechanism that has yet to be fully deciphered. Therefore, as a first step towards exploring the structural details of DRB3, we present a nearly complete backbone and side chain assignment of the two N-terminal dsRBD domains.

Key arginine residues in R2D2 dsRBD1 and dsRBD2 lead the siRNA recognition in Drosophila melanogaster RNAi pathway

In Drosophila melanogaster, Dcr-2:R2D2 heterodimer binds to the 21 nucleotide siRNA duplex to form the R2D2/Dcr-2 Initiator (RDI) complex, which is critical for the initiation of siRNA-induced silencing complex (RISC) assembly. During RDI complex formation, R2D2, a protein that contains three dsRNA binding domains (dsRBD), senses two aspects of the siRNA: thermodynamically more stable end (asymmetry sensing) and the 5′-phosphate (5'-P) recognition. Despite several detailed studies to date, the molecular determinants arising from R2D2 for performing these two tasks remain elusive. In this study, we have performed structural, biophysical, and biochemical characterization of R2D2 dsRBDs. We found that the solution NMR-derived structure of R2D2 dsRBD1 yielded a canonical α1-β1-β2-β3-α2 fold, wherein two arginine salt bridges provide additional stability to the R2D2 dsRBD1. Furthermore, we show that R2D2 dsRBD1 interacts with thermodynamically asymmetric siRNA duplex independent of its 5′-phosphorylation state, whereas R2D2 dsRBD2 prefers to interact with 5'-P siRNA duplex. The mutation of key arginine residues, R53 and R101, in concatenated dsRBDs of R2D2 results in a significant loss of siRNA duplex recognition. Our study deciphers the active roles of R2D2 dsRBDs by showing that dsRBD1 initiates siRNA recognition, whereas dsRBD2 senses 5′-phosphate as an authentic mark on functional siRNA.

Distinct domain organization and diversity of 2'-5'-oligoadenylate synthetases.

The 2'-5'-oligoadenylate synthetases (OAS) are important components of the innate immune system that recognize viral double-stranded RNA (dsRNA). Upon dsRNA binding, OAS generate 2'-5'-linked oligoadenylates (2-5A) that activate ribonuclease L (RNase L), halting viral replication. The OAS/RNase L pathway is thus an important antiviral pathway and viruses have devised strategies to circumvent OAS activation. OAS enzymes are divided into four classes according to size: small (OAS1), medium (OAS2), and large (OAS3) that consist of one, two, and three OAS domains, respectively, and the OAS-like protein (OASL) that consists of one OAS domain and tandem domains similar to ubiquitin. Early investigation of the OAS enzymes hinted at the recognition of dsRNA by OAS, but due to size differences amongst OAS family members combined with the lack of structural information on full-length OAS2 and OAS3, the regulation of OAS catalytic activity by dsRNA was not well understood. However, the recent biophysical studies of OAS have highlighted overall structure and domain organization. In this review, we present a detailed examination of the OAS literature and summarized the investigation on 2'-5'-oligoadenylate synthetases.

Abstract 3254: Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines

Abstract mRNA-based therapeutics have shown clinical efficacy, evidenced by the success of the mRNA vaccines for SARS-CoV-2. Personalized mRNA vaccines containing patient-derived tumor mutations are now entering the clinic and have shown promise. Delivery of mRNA-encoded therapeutic proteins (e.g., antibodies) is also under development. mRNA drug substance is typically generated by in vitro transcription (IVT). One byproduct and contaminant of IVT is the formation of double-stranded RNA (dsRNA). dsRNA is highly immunogenic, can induce inflammation and cell death, and inhibit protein translation, including the protein encoded by the therapeutic mRNA. As such, dsRNA must be measured for all IVT products to ensure safety and efficacy. However, existing dot bot and ELISA technology to detect dsRNA is neither sensitive nor quantitative. Herein, we describe the development of novel assays for sensitive and specific quantitation of dsRNA in IVT products and following encapsulation of IVT products in lipid nanoparticles (LNPs). First, NanoBiT® split luciferase technology was applied to detect dsRNA in IVT products. dsRNA binding domains were genetically attached to either SmBiT or LgBiT. In the presence of dsRNA, the SmBiT and LgBiT constructs bind via the dsRNA binding domains, resulting in dimerization and the complementation of functional NanoBiT® luciferase. NanoBiT® then generates light which is proportional to the amount of dsRNA in the sample. The assay detects dsRNA greater than or equal to 30 base pairs and is independent of the size and sequence of the dsRNA contaminant. The assay does not cross-react with single-stranded RNA or DNA and is highly sensitive, with a limit of detection less than 100 pg/ml. As a complementary tool to the biochemical assay, we developed a cell-based bioassay to detect dsRNA encapsulated in LNPs. dsRNA/LNPs are taken up by endocytosis where Toll-like receptor 3 (TLR3) detects dsRNA. Activation of TLR3 initiates a signaling pathway that is detected by a TLR3 pathway-specific promoter driving luciferase expression. This pathway likely represents the most physiologically-relevant readout of dsRNA bioactivity. Both assays are developed in “add-mix-read” format with no wash steps or media transfer, and exhibit robust, sensitive, and repeatable performance for specific detection of dsRNA in mixed solutions. Citation Format: Rich Moravec, Jun Wang, Jim Hartnett, Mei Cong, Jamison Grailer. Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3254.

Abstract 5448: Malignant RNA editing by inflammation-responsive RNA editase ADAR1 drives T-cell leukemia relapse via attenuating dsRNA sensing

Abstract Leukemia-initiating cells (LICs) play a crucial role in leukemia research and treatment, being considered the source of relapse and resistance, posing a substantial challenge for successful management of the disease. It's crucial to identify the key factors that drive LIC self-renewal to develop targeted methods for eliminating LICs and preventing relapse. In this study, we demonstrate that the RNA editing enzyme ADAR1 plays a vital role as a determinant of stemness, promoting LIC self-renewal by reducing the detection of abnormal double-stranded RNA (dsRNA). As a result, ADAR1-knockdown significantly impairs the ability of LICs to self-renew and extends survival in T-ALL PDX models. We performed RNA-seq studies on enriched T-ALL LICs (CD34+Lin-). The lentivirus-to-cell ratio was optimized to achieve a 50% reduction in ADAR1, resulting in differential expression of 661 genes, with downregulation of critical self-renewal genes in ADAR1-deficient leukemia-initiating cells. Importantly, additional data indicated that ADAR1 likely influences self-renewal gene expression independently of A-to-I RNA editing, revealing novel regulatory mechanisms in leukemia. Knockdown of MDA5 and ADAR1 in patient-derived xenograft (PDX) T-ALL leukemia-initiating cells (LICs) revealed diverse rescue effects on self-renewal in different PDX models. The differential response was associated with variations in gene expression patterns in Rig-I-Like signaling and cytosolic sensing pathways, suggesting intrinsic differences in IFN signaling properties among patients. Moreover, the study proposes a patient-specific reliance on the ADAR1 p150-MDA5 axis for promoting self-renewal, as evidenced by differential rescue effects and varying expression levels of ADAR1 isoforms and MDA5 in T-ALL patient samples and cell lines. ADAR1, beyond its role in RNA editing, acts as a dsRNA binding protein, exerting RNA editing-independent functions to suppress aberrant IFN signaling and promote cancer progression. Using Jurkat cells with high intrinsic MDA5 levels, an ADAR1 knockout (KO) was generated, revealing that ADAR1 is upregulated by IFN treatment, predominantly the p150 isoform. We explored the dual roles of ADAR1—RNA editing-dependent and -independent—in suppressing IFN-induced apoptosis, demonstrating the necessity of both for optimal function. Additionally, ADAR1 was found to suppress cytoplasmic dsRNA sensing pathways, such as RIG-I and PKR, and its RNA editing-independent activity contributes significantly to the suppression of interferon-stimulated gene (ISG) expression in T-ALL. In summary, our findings reveal that ADAR1 acts as a factor that promotes self-renewal while limiting the recognition of naturally occurring dsRNA. Therefore, targeting ADAR1 emerges as a safe and effective therapeutic approach for eradicating LICs in T-ALL. Citation Format: Maria Dolores Rivera, Haoran Zhang, Qingchen Zhou, Jessica Pham, Jane Isquith, Roman Sasik, Adam Mark, Sabina Enlund, Frida Holm, Ma Wenxue, Kathleen Fisch, Dennis Kuo, Catriona Jamieson, Qingfei Jiang. Malignant RNA editing by inflammation-responsive RNA editase ADAR1 drives T-cell leukemia relapse via attenuating dsRNA sensing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5448.

A double-stranded RNA binding protein enhances drought resistance via protein phase separation in rice

Drought stress significantly impacts global rice production, highlighting the critical need to understand the genetic basis of drought resistance in rice. Here, through a genome-wide association study, we reveal that natural variations in DROUGHT RESISTANCE GENE 9 (DRG9), encoding a double-stranded RNA (dsRNA) binding protein, contribute to drought resistance. Under drought stress, DRG9 condenses into stress granules (SGs) through liquid-liquid phase separation via a crucial α-helix. DRG9 recruits the mRNAs of OsNCED4, a key gene for the biosynthesis of abscisic acid, into SGs and protects them from degradation. In drought-resistant DRG9 allele, natural variations in the coding region, causing an amino acid substitution (G267F) within the zinc finger domain, increase DRG9’s binding ability to OsNCED4 mRNA and enhance drought resistance. Introgression of the drought-resistant DRG9 allele into the elite rice Huanghuazhan significantly improves its drought resistance. Thus, our study underscores the role of a dsRNA-binding protein in drought resistance and its promising value in breeding drought-resistant rice.

Genetic underpinnings of arthropod community distributions in Populus trichocarpa.

Community genetics seeks to understand the mechanisms by which natural genetic variation in heritable host phenotypes can encompass assemblages of organisms such as bacteria, fungi, and many animals including arthropods. Prior studies that focused on plant genotypes have been unable to identify genes controlling community composition, a necessary step to predict ecosystem structure and function as underlying genes shift within plant populations. We surveyed arthropods within an association population of Populus trichocarpa in three common gardens to discover plant genes that contributed to arthropod community composition. We analyzed our surveys with traditional single-trait genome-wide association analysis (GWAS), multitrait GWAS, and functional networks built from a diverse set of plant phenotypes. Plant genotype was influential in structuring arthropod community composition among several garden sites. Candidate genes important for higher level organization of arthropod communities had broadly applicable functions, such as terpenoid biosynthesis and production of dsRNA binding proteins and protein kinases, which may be capable of targeting multiple arthropod species. We have demonstrated the ability to detect, in an uncontrolled environment, individual genes that are associated with the community assemblage of arthropods on a host plant, further enhancing our understanding of genetic mechanisms that impact ecosystem structure.

RNA recognition by PKR during DNA virus infection.

Protein kinase R (PKR) is a double-stranded RNA (dsRNA) binding protein that plays a crucial role in innate immunity during viral infection and can restrict both DNA and RNA viruses. The potency of its antiviral function is further reflected by the large number of viral-encoded PKR antagonists. However, much about the regulation of dsRNA accumulation and PKR activation during viral infection remains unknown. Since DNA viruses do not have an RNA genome or RNA replication intermediates like RNA viruses do, PKR-mediated dsRNA detection in the context of DNA virus infection is particularly intriguing. Here, we review the current state of knowledge regarding the regulation of PKR activation and its antagonism during infection with DNA viruses.

Neofunctionalization driven by positive selection led to the retention of the loqs2 gene encoding an Aedes specific dsRNA binding protein

BackgroundMosquito borne viruses, such as dengue, Zika, yellow fever and Chikungunya, cause millions of infections every year. These viruses are mostly transmitted by two urban-adapted mosquito species, Aedes aegypti and Aedes albopictus. Although mechanistic understanding remains largely unknown, Aedes mosquitoes may have unique adaptations that lower the impact of viral infection. Recently, we reported the identification of an Aedes specific double-stranded RNA binding protein (dsRBP), named Loqs2, that is involved in the control of infection by dengue and Zika viruses in mosquitoes. Preliminary analyses suggested that the loqs2 gene is a paralog of loquacious (loqs) and r2d2, two co-factors of the RNA interference (RNAi) pathway, a major antiviral mechanism in insects.ResultsHere we analyzed the origin and evolution of loqs2. Our data suggest that loqs2 originated from two independent duplications of the first double-stranded RNA binding domain of loqs that occurred before the origin of the Aedes Stegomyia subgenus, around 31 million years ago. We show that the loqs2 gene is evolving under relaxed purifying selection at a faster pace than loqs, with evidence of neofunctionalization driven by positive selection. Accordingly, we observed that Loqs2 is localized mainly in the nucleus, different from R2D2 and both isoforms of Loqs that are cytoplasmic. In contrast to r2d2 and loqs, loqs2 expression is stage- and tissue-specific, restricted mostly to reproductive tissues in adult Ae. aegypti and Ae. albopictus. Transgenic mosquitoes engineered to express loqs2 ubiquitously undergo developmental arrest at larval stages that correlates with massive dysregulation of gene expression without major effects on microRNAs or other endogenous small RNAs, classically associated with RNA interference.ConclusionsOur results uncover the peculiar origin and neofunctionalization of loqs2 driven by positive selection. This study shows an example of unique adaptations in Aedes mosquitoes that could ultimately help explain their effectiveness as virus vectors.

The SIDT2/MDA5/IFN axis contributes to virus resistance in teleost fish

Disease resistance varies among fish species. The genetic factors contributing to disease resistance are largely unknown. Here, by comparative analysis on the disease resistance trait of grass carp (Ctenopharyngodon idellus, Ci) and barbel chub (Squaliobarbus curriculus, Sc), we report that the genes encoding SIDT2, MDA5 and IFN are associated with the resistance to viral infection. We identified the amino acid difference of SIDT2s, the existing or lacking of dsRNA binding loci in the carboxy-terminal domain (CTD) of MDA5s, and the promoter difference of ifn2s between the two fish species. Both CiSIDT2 and ScSIDT2 negatively regulate the MDA5-IRF3/7 mediated type I IFN reaction, and ScSIDT2 exhibited a stronger ability than CiSIDT2 to induce Ciirf7, Ciifn1, Ciifn2 and Cimx expressions, and to resist GCRV infection. ScMDA5 showed a greater ability than CiMDA5 in the induction of IRF7-mediated type I interferon and anti-GCRV function. CiMDA5 CTD inhibited Ciifn1 while enhanced Ciifn2 exrpessions, and ScMDA5 CTD exerts opposite effects on Ciifn1 and Ciifn2 expressions. ScMDA5 CTD possesses the ability to bind with GCRV nucleic acid fragments, while CiMDA5 lacking binding with virus genome. The promoter activities of Ciifn2 is significantly higher than that of Scifn2 owning to their differences in promoter sequences and binding proteins. These data revealed the mechanism of SIDT2-MDA5-IFN genetic architecture contributing to the difference in interspecies resistance and opening the possibility of molecular breeding for fish resistance.