dsRNA Binding Research Articles

Characterization and functional analysis of chicken dsRNA binding protein hnRNPU

In mammals, heterogeneous ribonucleoprotein U (hnRNPU), also named as nuclear matrix protein-nuclear scaffold attachment factor (SAFA), was originally identified as a DNA/RNA interactor protein. It has been reported that human hnRNPU facilitates IFN-β generation after vesicular stomatitis virus (VSV) infection. Nevertheless, the role of chicken hnRNPU (chhnRNPU) in IFN-β regulation as well as in infectious bursal diseases virus (IBDV) replication is still unclear. Here, we found that chhnRNPU inhibits IFN-β production via interacting with MDA5 and MAVS, and facilitates IBDV replication via associating with genomic dsRNA of IBDV. Firstly, chicken hnRNPU (chhnRNPU) was widely expressed in different tissues of chickens and was distributed in the nucleus of DF-1 cells. Overexpression of chhnRNPU significantly suppresses IFN-β promoter activities induced by MDA5 and MAVS. Additionally, immunoprecipitated by dsRNA antibodies, which followed LC-MS analysis demonstrate that chhnRNPU is a partner of viral genomic dsRNA. chhnRNPU is translocated from nucleus to cytosol to co-localize with replication complex of IBDV after IBDV infection. Over-expression of chhnRNPU significantly promotes IBDV replication, which was determined by western blotting, qRT-PCR and TCID50 assay. Furthermore, knock down chhnRNPU by siRNA remarkably facilitates IFN-β production, and inhibits IBDV proliferation. These data collectively reveal that chhnRNPU positively regulates IBDV replication via negatively regulating IFN-β response.

Triphenylphosphine gold(I) derivatives promote antiviral effects against the Chikungunya virus.

Herein a systematic series of four [AuLL']n+ n=0, +1 complexes, where L=1,3-bis(mesityl)imidazole-2-ylidene (IMes), or triphenylphosphine (PPh3), and L' = chloride, or 4-dimethylaminopyridine (DMAP), had their in vitro antiviral activity assessed against Chikungunya virus (CHIKV). The PPh3 derivatives inhibited viral replication by 99%, whereas the IMes derivatives about 50%. The lipophilicity of the PPh3 derivatives is higher than the IMes-bearing compounds, which can be related to their more prominent antiviral activities. The dissociation of DMAP is faster than chloride in solution for both IMes and PPh3 derivatives; however, it does not significantly affect their in vitro activities, showing a higher dependence on the nature of L rather than L' towards their antiviral effects. All complexes bind to N-acetyl-L-cysteine, with the Ph3P-bearing complexes coordinating at a faster rate to this amino acid. The binding constants to bovine serum albumin are in the order of 104, slightly higher for the DMAP complexes in both PPh3 and IMes derivatives. Mechanistic investigations of the PPh3 complexes showed a ubiquitous protective effect of the compounds in the pretreatment, early stages, and post-entry assays. The most significant inhibition was observed in post-entry activity, in which the complexes blocked viral replication in 99%, followed by up to 95% inhibition of the early stages of infection. Pretreatment assays showed a 92% and 80% replication decrease for the chloride and DMAP derivatives, respectively. dsRNA binding assays showed a significant interaction of the compounds with dsRNA, an essential biomolecule to viral replication.

Crocodilepox Virus Protein 157 Is an Independently Evolved Inhibitor of Protein Kinase R

Crocodilepox virus (CRV) belongs to the Poxviridae family and mainly infects hatchling and juvenile Nile crocodiles. Most poxviruses encode inhibitors of the host antiviral protein kinase R (PKR), which is activated by viral double-stranded (ds) RNA formed during virus replication, resulting in the phosphorylation of eIF2α and the subsequent shutdown of general mRNA translation. Because CRV lacks orthologs of known poxviral PKR inhibitors, we experimentally characterized one candidate (CRV157), which contains a predicted dsRNA-binding domain. Bioinformatic analyses indicated that CRV157 evolved independently from other poxvirus PKR inhibitors. CRV157 bound to dsRNA, co-localized with PKR in the cytosol, and inhibited PKR from various species. To analyze whether CRV157 could inhibit PKR in the context of a poxvirus infection, we constructed recombinant vaccinia virus strains that contain either CRV157, or a mutant CRV157 deficient in dsRNA binding in a strain that lacks PKR inhibitors. The presence of wild-type CRV157 rescued vaccinia virus replication, while the CRV157 mutant did not. The ability of CRV157 to inhibit PKR correlated with virus replication and eIF2α phosphorylation. The independent evolution of CRV157 demonstrates that poxvirus PKR inhibitors evolved from a diverse set of ancestral genes in an example of convergent evolution.

Structural insights into dsRNA processing by Drosophila Dicer-2–Loqs-PD

Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes5,6. Here we report the cryo-electron microscopy structures of Dcr-2–Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5′-phosphate-binding pocket. The overall conformation of Dcr-2–Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2–Loqs-PD.

MERS-CoV endoribonuclease and accessory proteins jointly evade host innate immunity during infection of lung and nasal epithelial cells

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into humans in 2012, causing highly lethal respiratory disease. The severity of disease may be, in part, because MERS-CoV is adept at antagonizing early innate immune pathways—interferon (IFN) production and signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L)—activated in response to viral double-stranded RNA (dsRNA) generated during genome replication. This is in contrast to severe acute respiratory syndrome CoV-2 (SARS-CoV-2), which we recently reported to activate PKR and RNase L and, to some extent, IFN signaling. We previously found that MERS-CoV accessory proteins NS4a (dsRNA binding protein) and NS4b (phosphodiesterase) could weakly suppress these pathways, but ablation of each had minimal effect on virus replication. Here we investigated the antagonist effects of the conserved coronavirus endoribonuclease (EndoU), in combination with NS4a or NS4b. Inactivation of EndoU catalytic activity alone in a recombinant MERS-CoV caused little if any effect on activation of the innate immune pathways during infection. However, infection with recombinant viruses containing combined mutations with inactivation of EndoU and deletion of NS4a or inactivation of the NS4b phosphodiesterase promoted robust activation of dsRNA-induced innate immune pathways. This resulted in at least tenfold attenuation of replication in human lung–derived A549 and primary nasal cells. Furthermore, replication of these recombinant viruses could be rescued to the level of wild-type MERS-CoV by knockout of host immune mediators MAVS, PKR, or RNase L. Thus, EndoU and accessory proteins NS4a and NS4b together suppress dsRNA-induced innate immunity during MERS-CoV infection in order to optimize viral replication.

Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly.

Ebola virus (EBOV) VP35 is a polyfunctional protein involved in viral genome packaging, viral polymerase function, and host immune antagonism. The mechanisms regulating VP35's engagement in different functions are not well-understood. We previously showed that the host E3 ubiquitin ligase TRIM6 ubiquitinates VP35 at lysine 309 (K309) to facilitate virus replication. However, how K309 ubiquitination regulates the function of VP35 as the viral polymerase co-factor and the precise stage(s) of the EBOV replication cycle that require VP35 ubiquitination are not known. Here, we generated recombinant EBOVs encoding glycine (G) or arginine (R) mutations at VP35/K309 (rEBOV-VP35/K309G/-R) and show that both mutations prohibit VP35/K309 ubiquitination. The K309R mutant retains dsRNA binding and efficient type-I Interferon (IFN-I) antagonism due to the basic residue conservation. The rEBOV-VP35/K309G mutant loses the ability to efficiently antagonize the IFN-I response, while the rEBOV-VP35/K309R mutant's suppression is enhanced. The replication of both mutants was significantly attenuated in both IFN-competent and -deficient cells due to impaired interactions with the viral polymerase. The lack of ubiquitination on VP35/K309 or TRIM6 deficiency disrupts viral transcription with increasing severity along the transcriptional gradient. This disruption of the transcriptional gradient results in unbalanced viral protein production, including reduced synthesis of the viral transcription factor VP30. In addition, lack of ubiquitination on K309 results in enhanced interactions with the viral nucleoprotein and premature nucleocapsid packaging, leading to dysregulation of virus assembly. Overall, we identified a novel role of VP35 ubiquitination in coordinating viral transcription and assembly.

A cryptic pocket in Ebola VP35 allosterically controls RNA binding

Protein-protein and protein-nucleic acid interactions are often considered difficult drug targets because the surfaces involved lack obvious druggable pockets. Cryptic pockets could present opportunities for targeting these interactions, but identifying and exploiting these pockets remains challenging. Here, we apply a general pipeline for identifying cryptic pockets to the interferon inhibitory domain (IID) of Ebola virus viral protein 35 (VP35). VP35 plays multiple essential roles in Ebola’s replication cycle but lacks pockets that present obvious utility for drug design. Using adaptive sampling simulations and machine learning algorithms, we predict VP35 harbors a cryptic pocket that is allosterically coupled to a key dsRNA-binding interface. Thiol labeling experiments corroborate the predicted pocket and mutating the predicted allosteric network supports our model of allostery. Finally, covalent modifications that mimic drug binding allosterically disrupt dsRNA binding that is essential for immune evasion. Based on these results, we expect this pipeline will be applicable to other proteins.

Enhanced replication fitness of MERS-CoV clade B over clade A strains in camelids explains the dominance of clade B strains in the Arabian Peninsula

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) continues infecting humans and dromedary camels. While MERS-CoV strains from the Middle East region are subdivided into two clades (A and B), all the contemporary epidemic viruses belong to clade B. Thus, MERS-CoV clade B strains may display adaptive advantages over clade A in humans and/or reservoir hosts. To test this hypothesis in vivo, we compared an early epidemic clade A strain (EMC/2012) with a clade B strain (Jordan-1/2015) in an alpaca model monitoring virological and immunological parameters. Further, the Jordan-1/2015 strain has a partial amino acid (aa) deletion in the double-stranded (ds) RNA binding motif of the open reading frame ORF4a protein. Animals inoculated with the Jordan-1/2015 variant had higher MERS-CoV replicative capabilities in the respiratory tract and larger nasal viral shedding. In the nasal mucosa, the Jordan-1/2015 strain caused an early IFN response, suggesting a role for ORF4a as a moderate IFN antagonist in vivo. However, both strains elicited maximal transcription of antiviral interferon-stimulated genes (ISGs) at the peak of infection on 2 days post inoculation, correlating with subsequent decreases in tissular viral loads. Genome alignment analysis revealed several clade B-specific amino acid substitutions occurring in the replicase and the S proteins, which could explain a better adaptation of clade B strains in camelid hosts. Differences in replication and shedding reported herein indicate a better fitness and transmission capability of MERS-CoV clade B strains than their clade A counterparts.

ILF3 represses repeat-derived microRNAs targeting RIG-I mediated type I interferon response

MicroRNAs (miRNAs) play important roles in regulated gene expression and miRNA biogenesis is also subject to regulation, together constituting critical regulatory circuitries in numerous physiological and pathological processes. As a dsRNA binding protein, interleukin enhancer binding factor 3 (ILF3) has been implicated as a negative regulator in miRNA biogenesis, but the mechanism and specificity have remained undefined. Here, combining small-RNA-seq and CLIP-seq, we showed that ILF3 directly represses many miRNAs or perhaps other types of small RNAs annotated in both miRBase and MirGeneDB. We demonstrated that ILF3 preferentially binds to A/U-enriched motifs, which tend to lengthen and/or stabilize the stem-loop in pri-miRNAs, thereby effectively competing with the Microprocessor to block miRNA biogenesis. Focusing on the biological function of ILF3-suppressed miR-582-3p, we discovered that this LINE-derived miRNA targets a critical interferon-inducible gene RIG-I for repression, thus establishing a novel ILF3/miR-582/RIG-I axis in the antiviral response.

Non-Cationic RGD-Containing Protein Nanocarrier for Tumor-Targeted siRNA Delivery.

Despite the recent successes in siRNA therapeutics, targeted delivery beyond the liver remains the major hurdle for the widespread application of siRNA in vivo. Current cationic liposome or polymer-based delivery agents are restricted to the liver and suffer from off-target effects, poor clearance, low serum stability, and high toxicity. In this study, we genetically engineered a non-cationic non-viral tumor-targeted universal siRNA nanocarrier (MW 26 KDa). This protein nanocarrier consists of three function domains: a dsRNA binding domain (dsRBD) (from human protein kinase R) for any siRNA binding, 18-histidine for endosome escape, and two RGD peptides at the N- and C-termini for targeting tumor and tumor neovasculature. We showed that cloned dual-RGD-dsRBD-18his (dual-RGD) protein protects siRNA against RNases, induces effective siRNA endosomal escape, specifically targets integrin αvβ3 expressing cells in vitro, and homes siRNA to tumors in vivo. The delivered siRNA leads to target gene knockdown in the cell lines and tumor xenografts with low toxicity. This multifunctional and biomimetic siRNA carrier is biodegradable, has low toxicity, is suitable for mass production by fermentation, and is serum stable, holding great potential to provide a widely applicable siRNA carrier for tumor-targeted siRNA delivery.

MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.

Modulation of MicroRNA Processing by Dicer via Its Associated dsRNA Binding Proteins.

MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR).

Dissecting the Role of N-Glycan at N413 in Toll-like Receptor 3 via Molecular Dynamics Simulations.

Toll-like receptor 3 (TLR3) is an endosomal receptor involved in initiating immune responses upon viral infection by directly recognizing double-stranded RNA (dsRNA). As one of the most heavily glycosylated TLR family members, the role of glycan at N413 of TLR3 in ligand recognition has been in debate for decades. Herein, to investigate the role of glycans in TLR3, specifically at amino acid residue N413, molecular dynamic simulations were performed. The loop region of LRR12 (residues 323-355), which protrudes from the dsRNA binding TLR3 lateral surface was found to be vital for interacting with dsRNA via the formation of hydrogen bonds. The glycan at N413 not only prevented dsRNA from being exposed to the bulk water during the binding process but further stabilized dsRNA in the TLR3 binding site. When N413 was in the glycosylated form, the binding free energy of TLR3 interacting with dsRNA was significantly lower than that of TLR3 in the N413 unglycosylated form. Additionally, as the glycan at N413 functioned to alter the dynamics of the dsRNA binding process, its flexibility was meanwhile influenced by dsRNA. In all, these results demonstrate that the size, length, and branch of glycan at N413 affect the thermodynamics and dynamics of TLR3 recognition with dsRNA. This study further extends our understanding of the biological role of glycans in the innate immune recognition of dsRNA by TLR3 and provides a new perspective for modulating TLR3 function.

Presence of two RIG-I-like receptors, MDA5 and LGP2, and their dsRNA binding capacity in a perciform fish, the snakehead Channa argus

Fish retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are critical RNA sensors in cytoplasm and are involved in antiviral innate immunity. However, some species of fish lack RIG-I gene, and the function of RLR members in RIG-I-absent fish is poorly understood. In the present study, MDA5, LGP2 and MAVS genes were identified in commercially important snakehead Channa argus. But, RIG-I gene was not found in this fish, and a systematic analysis of RLRs in available genome database of fish indicated the absence of RIG-I in the Acanthomorphata, Clupeiformes and Polypteriformes, suggesting that loss events of RIG-I gene may have occurred independently three times in the evolutionary history of fish. The MDA5, LGP2 and MAVS in snakehead have conserved protein domains and genomic location based on sequence, phylogenetic and syntenic analyses. These genes are constitutively expressed in healthy fish and can be induced by polyinosinic and polycytidylic acid (poly(I:C)) stimulation in vitro. It is further revealed that the snakehead MDA5 and LGP2 have binding capacity with dsRNA, such as poly(I:C), and MDA5 can interact with MAVS, implying the antiviral function of MDA5 in the RIG-I-absent fish.

Murine Norovirus Infection Results in Anti-inflammatory Response Downstream of Amino Acid Depletion in Macrophages.

ABSTRACTMurine norovirus (MNV) infection results in a late translation shutoff that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the α subunit of eukaryotic initiation factor 2 (eIF2α) kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signaling during metabolic stress. While viral infection is usually associated with activation of double-stranded RNA (dsRNA) binding pattern recognition receptor PKR, we hypothesized that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterize cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signaling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation.IMPORTANCE During viral infection, host defenses are typically characterized by the secretion of proinflammatory autocrine and paracrine cytokines, potentiation of the interferon (IFN) response, and induction of the antiviral response via activation of JAK and Stat signaling. To avoid these and propagate, viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signaling. This highlights novel tools in the viral countermeasures arsenal and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis.

Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.

The multi-functional reovirus σ3 protein is a virulence factor that suppresses stress granule formation and is associated with myocardial injury.

The mammalian orthoreovirus double-stranded (ds) RNA-binding protein σ3 is a multifunctional protein that promotes viral protein synthesis and facilitates viral entry and assembly. The dsRNA-binding capacity of σ3 correlates with its capacity to prevent dsRNA-mediated activation of protein kinase R (PKR). However, the effect of σ3 binding to dsRNA during viral infection is largely unknown. To identify functions of σ3 dsRNA-binding activity during reovirus infection, we engineered a panel of thirteen σ3 mutants and screened them for the capacity to bind dsRNA. Six mutants were defective in dsRNA binding, and mutations in these constructs cluster in a putative dsRNA-binding region on the surface of σ3. Two recombinant viruses expressing these σ3 dsRNA-binding mutants, K287T and R296T, display strikingly different phenotypes. In a cell-type dependent manner, K287T, but not R296T, replicates less efficiently than wild-type (WT) virus. In cells in which K287T virus demonstrates a replication deficit, PKR activation occurs and abundant stress granules (SGs) are formed at late times post-infection. In contrast, the R296T virus retains the capacity to suppress activation of PKR and does not mediate formation of SGs at late times post-infection. These findings indicate that σ3 inhibits PKR independently of its capacity to bind dsRNA. In infected mice, K287T produces lower viral titers in the spleen, liver, lungs, and heart relative to WT or R296T. Moreover, mice inoculated with WT or R296T viruses develop myocarditis, whereas those inoculated with K287T do not. Overall, our results indicate that σ3 functions to suppress PKR activation and subsequent SG formation during viral infection and that these functions correlate with virulence in mice.

110-OR: MDA5-Dependent Responses Can Influence Spontaneous and Virus-Accelerated T1D Onset

Coxsackievirus B3 (CVB3) infection promotes the activation of proinflammatory immune cells and is associated with triggering type 1 diabetes (T1D). We previously demonstrated that CVB3 infection of Non-Obese Diabetic (NOD) mice accelerates T1D, partly due to the activation of melanoma differentiation-associated protein 5 (MDA5)-dependent antiviral responses including IFN-α/β synthesis. Mutations within human IFIH1, the gene encoding MDA5, are correlated with T1D risk. Yet, the role of MDA5 on immune cells during T1D remains unknown. By utilizing mice that lack (KO) or contain an in-frame deletion within the helicase 1 domain of MDA5 (ΔHel1), we can test the hypothesis that reduced MDA5 responses delay T1D by decreasing IFN-α/β synthesis and immune cell activation. NOD and KO mice develop T1D similarly, but ΔHel1 mice have delayed spontaneous and CVB3-accelerated T1D. At 12 weeks of age during spontaneous T1D, ΔHel1 mice have a significant reduction in IFNγ+ CD4+ T cells (6.2-fold), perforin+ CD8+ T cells (13.2-fold), and TNF+ F4/80+ macrophages (MΦ) (1.7-fold) compared to NOD and KO mice. CVB3-infected ΔHel1 mice at day 3 exhibited a significant decrease in pancreatic IFN-α (2.2-fold) and IFN-β (3.1-fold) synthesis compared to NOD mice. At day 7 post-infection, ΔHel1 mice had a significant reduction in pancreatic TNF+ F4/80+ MΦ (2-fold) compared to NOD and KO mice. ΔHel1 and KO mice had reduced pancreatic IFNγ+ CD4+ T cells (1.9-fold) and perforin+ CD8+ T cells (2.6-fold) compared to NOD mice following CVB3 infection. This data demonstrated that the ΔHel1 mutation elicits a decrease in basal and CVB3-responsive MΦ and T cells within the pancreata to delay T1D. However, KO mice have impaired T cell responses to CVB3 and failed to delay spontaneous and CVB3-accelerated T1D indicating MDA5-dependent responses may be necessary for tolerance induction. Future studies will examine the mechanism of ΔHel1 T1D protection and whether MDA5 ATPase activity and/or dsRNA binding can modulate T1D development. Disclosure S. I. Blum: None. Y. Chen: None. H. M. Tse: None. Funding DK099550, DK127497, DK1264565, T32GM1097805, T32AI007051

K160 in the RNA-binding domain of the orf virus virulence factor OV20.0 is critical for its functions in counteracting host antiviral defense.

The OV20.0 virulence factor of orf virus antagonizes host antiviral responses. One mechanism through which it functions is by inhibiting activation of the dsRNA-activated protein kinase R (PKR) by sequestering dsRNA and by physically interacting with PKR. Sequence alignment indicated that several key residues critical for dsRNA binding were conserved in OV20.0, and their contribution to OV20.O function was investigated in this study. We found that residues F141, K160, and R164 were responsible for the dsRNA-binding ability of OV20.0. Interestingly, mutation at K160 (K160A) diminished the OV20.0-PKR interaction and further reduced the inhibitory effect of OV20.0 on PKR activation. Nevertheless, OV20.0 homodimerization was not influenced by K160A. The contribution of the dsRNA-binding domain and K160 to the suppression of RNA interference by OV20.0 was further demonstrated in plants. In summary, K160 is essential for the function of OV20.0, particularly its interaction with dsRNA and PKR that ultimately contributes to the suppression of PKR activation.

Analysis of Porcine RIG-I Like Receptors Revealed the Positive Regulation of RIG-I and MDA5 by LGP2.

The RLRs play critical roles in sensing and fighting viral infections especially RNA virus infections. Despite the extensive studies on RLRs in humans and mice, there is a lack of systemic investigation of livestock animal RLRs. In this study, we characterized the porcine RLR members RIG-I, MDA5 and LGP2. Compared with their human counterparts, porcine RIG-I and MDA5 exhibited similar signaling activity to distinct dsRNA and viruses, via similar and cooperative recognitions. Porcine LGP2, without signaling activity, was found to positively regulate porcine RIG-I and MDA5 in transfected porcine alveolar macrophages (PAMs), gene knockout PAMs and PK-15 cells. Mechanistically, LGP2 interacts with RIG-I and MDA5 upon cell activation, and promotes the binding of dsRNA ligand by MDA5 as well as RIG-I. Accordingly, porcine LGP2 exerted broad antiviral functions. Intriguingly, we found that porcine LGP2 mutants with defects in ATPase and/or dsRNA binding present constitutive activity which are likely through RIG-I and MDA5. Our work provided significant insights into porcine innate immunity, species specificity and immune biology.