RIG-I-like Receptor Pathway Research Articles

TRIM103 activates the RLRs pathway to enhance antiviral response by targeting VP5 and VP7

Grass carp (Ctenopharyngodon idella), crucial to global inland aquaculture with a production of 5.8 million tones in 2020, faces significant challenges from hemorrhagic disease caused by grass carp reovirus (GCRV). Rapid mutations compromise current vaccines, underscoring the need for a deeper understanding of antiviral mechanisms to enhance molecular marker-assisted selection. This study investigates the role of Tripartite Motif (TRIM) family in the innate immune response of grass carp, focusing on TRIM103 from Ctenopharyngodon Idella (CiTRIM103), a member of the TRIM-B30.2 family, which includes proteins with the B30.2 domain at the N-terminus, known for antiviral properties in teleosts. CiTRIM103 bind to the outer coat proteins VP5 and VP7 of GCRV. This binding is theorized to strengthen the function of the RIG-I-like Receptor (RLR) signaling pathway, crucial for antiviral responses. Demonstrations using overexpression and RNA interference (RNAi) techniques have shown that CiTRIM103 effectively inhibits GCRV replication. Moreover, molecular docking and pulldown assays suggest potential binding interactions of CiTRIM103's B30.2 domain with GCRV outer coat proteins VP5 and VP7. These interactions impede viral replication, enhance RLR receptor expression, and activate key transcription factors to induce type I interferons (IFNs). These findings elucidate the antiviral mechanisms of CiTRIM103, provide a foundation for future Molecular genetic breeding in grass carp.

Effects of Isochlorogenic Acid A on Mitochondrial Dynamics Imbalance and RLR Damage in PAM Cells Induced by Combined Mycotoxins

Mycotoxins have strong immunotoxicity and can induce oxidative stress and mitochondrial dynamics imbalance. Mitochondrial antiviral signaling protein (MAVS) in the RIG-I like receptor (RLR) pathway of innate immunity is located on mitochondria, and whether it is affected by mycotoxins has not been reported yet. This experiment used porcine alveolar macrophages (PAM) to evaluate the antagonism of three isomers of chlorogenic acid (chlorogenic acid, isochlorogenic acid A, and neochlorogenic acid) against combined mycotoxins (Aflatoxin B1, Deoxynivalenol, and Ochratoxin A) induced mitochondrial damage and their effects on the RLR pathway, providing assistance for further elucidating the mechanism of mycotoxin immunotoxicity. Western blotting, enzyme linked immunosorbent assay (ELISA), and flow cytometry were used to detect relevant indicators. All three types of chlorogenic acid treatment can antagonize the cytotoxicity induced by combined mycotoxins, especially isochlorogenic acid A, which can protect cells from mycotoxins damage by maintaining mitochondrial dynamic homeostasis and improving innate immune function related to the RLR pathway.

The RLR intrinsic antiviral system is expressed in neural retina and restricts lentiviral transduction of human Mueller cells

The retinoic acid-inducible gene I (RIG)-I-like receptor (RLR) family of RNA sensor proteins plays a key role in the innate immune response to viral nucleic acids, including viral gene delivery vectors, but little is known about the expression of RLR proteins in the retina. The purpose of this study was to characterize cell-specific expression patterns of RLR proteins in non-human primate (NHP) neural retina tissue and to examine if RLR pathway signaling restricts viral gene delivery transduction. Since RLR protein signaling converges at the mitochondrial antiviral signaling protein (MAVS), experiments were performed to determine if knockdown of MAVS affected FIVGFP transduction efficiency in the human Mueller cell line MIO-M1. Immunoblotting confirmed expression of RIG-I, melanoma differentiation-associated protein 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), and MAVS proteins in MIO-M1 cells and NHP retina tissue. Double label immunofluorescence (IF) studies revealed RIG-I, LGP2, and MAVS were expressed in Mueller microglial cells in the NHP retina. In addition, LGP2 and MDA5 proteins were detected in cone and retinal ganglion cells (RGC). MDA5 was also present in a subset of calretinin positive amacrine cells, and in nuclei within the inner nuclear layer (INL). Knockdown of MAVS significantly increased the transduction efficiency of the lentiviral vector FIVGFP in MIO-M1 cells, compared to control cells. FIVGFP or AAVGFP challenge did not alter expression of the LGP2, MAVS, MDA5 or RIG-I genes in MIO-M1 cells or NHP retina tissue compared to media treated controls. Our data demonstrate that innate immune response proteins involved in viral RNA sensing, including MDA5, RIG-I, LGP2, and MAVS, are expressed in several cell types within the NHP neural retina. In addition, the MAVS protein restricts non-human lentiviral transduction efficiency in MIO-M1 cells.

Clinical and genetic characterization of Epstein-Barr virus–associated T/NK-cell lymphoproliferative diseases

Epstein-Barr virus (EBV)-associated T-/natural killer (T/NK)-cell lymphoproliferative diseases clinically take on various forms, ranging from an indolent course to an aggressive condition. Clinically, failure to establish precise diagnosis and provide proper treatment makes it difficult to help patients. We sought to better understand the underlying pathogenesis and to identify genetic prognostic factors to achieve better treatment efficacy. In this study, 119 cases of EBV-associated lymphoproliferative diseases, including EBV-associated hemophagocytic lymphohistiocytosis (n= 46) and chronic active EBV disease of T/NK cell type (n= 73), were retrospectively examined. Adults aged >20 years at onset accounted for 71.4% of our cohort. About 54.6% patients with unfavorable overall survival developed hemophagocytic lymphohistiocytosis and had higher plasma EBV load. Allogenic hematopoietic stem-cell transplantation was the sole independent favorable factor. We systematically screened germline and somatic aberrations by whole-exome and targeted sequencing. Among 372 antiviral immunity genes, germline variants of 8 genes were significantly enriched. From a panel of 24 driver genes, somatic mutations were frequently identified in dominant EBV-infected T/NK cells. Patients carrying any germline/somatic aberrations in epigenetic modifiers and RIG-I-like receptor (RLR) pathway had worse overall survival than those without 2 type aberrations. Importantly, patients with IFIH1 and/or DDX3X aberrations in the RLR pathway had higher plasma and NK-cell EBV load. Knockdown of DDX3X in NKYS cells downregulated RLR signaling activities and elevated the expression of EBV-encoded oncogenes such as LMP1 and EBNA1. Genetic defects were prevalent in adult EBV-associated hemophagocytic lymphohistiocytosis patients and patients with chronic active EBV disease of T/NK cell type; these defects were associated with unfavorable prognosis. These findings can help clinicians work out more precise staging of the condition and provide new insights into these EBV-associated diseases.

Stress granules are shock absorbers that prevent excessive innate immune responses to dsRNA

Proper defense against microbial infection depends on the controlled activation of the immune system. This is particularly important for the RIG-I-like receptors (RLRs), which recognize viral dsRNA and initiate antiviral innate immune responses with the potential of triggering systemic inflammation and immunopathology. Here, we show that stress granules (SGs), molecular condensates that form in response to various stresses including viral dsRNA, play key roles in the controlled activation of RLR signaling. Without the SG nucleators G3BP1/2 and UBAP2L, dsRNA triggers excessive inflammation and immune-mediated apoptosis. In addition to exogenous dsRNA, host-derived dsRNA generated in response to ADAR1 deficiency is also controlled by SG biology. Intriguingly, SGs can function beyond immune control by suppressing viral replication independently of the RLR pathway. These observations thus highlight the multi-functional nature of SGs as cellular "shock absorbers" that converge on protecting cell homeostasis by dampening both toxic immune response and viral replication.

Different Mechanisms Are Utilized by Coronavirus Transmissible Gastroenteritis Virus To Regulate Interferon Lambda 1 and Interferon Lambda 3 Production.

Type III interferons (IFN-λ) are shown to be preferentially produced by epithelial cells, which provide front-line protection at barrier surfaces. Transmissible gastroenteritis virus (TGEV), belonging to the genus Alphacoronavirus of the family Coronaviridae, can cause severe intestinal injuries in porcine, resulting in enormous economic losses for the swine industry, worldwide. Here, we demonstrated that although IFN-λ1 had a higher basal expression, TGEV infection induced more intense IFN-λ3 production in vitro and in vivo than did IFN-λ1. We explored the underlying mechanism of IFN-λ induction by TGEV and found a distinct regulation mechanism of IFN-λ1 and IFN-λ3. The classical RIG-I-like receptor (RLR) pathway is involved in IFN-λ3 but not IFN-λ1 production. Except for the signaling pathways mediated by RIG-I and MDA5, TGEV nsp1 induces IFN-λ1 and IFN-λ3 by activating NF-κB via the unfolded protein responses (UPR) PERK-eIF2α pathway. Furthermore, functional domain analysis indicated that the induction of IFN-λ by the TGEV nsp1 protein was located at amino acids 85 to 102 and was dependent on the phosphorylation of eIF2α and the nuclear translocation of NF-κB. Moreover, the recombinant TGEV with the altered amino acid motif of nsp1 85-102 was constructed, and the nsp1 (85-102sg) mutant virus significantly reduced the production of IFN-λ, compared with the wild strain. Compared to the antiviral activities of IFN-λ1, the administration of IFN-λ3 showed greater antiviral activity against TGEV infections in IPEC-J2 cells. In summary, our data point to the significant role of IFN-λ in the host innate antiviral responses to coronavirus infections within mucosal organs and in the distinct mechanisms of IFN-λ1 and IFN-λ3 regulation. IMPORTANCE Coronaviruses cause infectious diseases in various mammals and birds and exhibit an epithelial cell tropism in enteric and respiratory tracts. It is critical to explore how coronavirus infections modulate IFN-λ, a key innate cytokine against mucosal viral infection. Our results uncovered the different processes of IFN-λ1 and IFN-λ3 production that are involved in the classical RLR pathway and determined that TGEV nsp1 induces IFN-λ1 and IFN-λ3 production by activating NF-κB via the PERK-eIF2α pathway in UPR. These studies highlight the unique regulation of antiviral defense in the intestine during TGEV infection. We also demonstrated that IFN-λ3 induced greater antiviral activity against TGEV replication than did IFN-λ1 in IPEC-J2 cells, which is helpful in finding a novel strategy for the treatment of coronavirus infections.

Bovine coronavirus nucleocapsid suppresses IFN-β production by inhibiting RIG-I-like receptors pathway in host cells.

The present study aimed to explore if bovine coronavirus nucleocapsid (BCoV N) impacts IFN-β production in the host cells and to reveal further molecular mechanism of BCoV pathogenesis. Human embryonic kidney (HEK) 293 T cells were transiently transfected with pMyc-BCoV-N recombinant plasmids, then infected with the vesicular stomatitis virus (VSV). Expression levels of beta interferon (IFN-β) mRNA were detected using RT-qPCR. The results showed that BCoV N gene was 1347 bp that was consistent with the expected size. pMyc-BCoV-N recombinant protein was 1347 bp which was successfully transcribed and overexpressed in HEK 293 T cells. BCoV-N recombinant protein inhibited dose-dependently VSV-induced IFN-β production (p < 0.01). MDA5, MAVS, TBK1 and IRF3 could promote transcription levels of IFN-β mRNA. But, BCoV-N protein demoted IFN-β transcription levels induced by MDA5, MAVS, TBK1 and IRF3. Furthermore, expression levels of MDA5, MAVS, TBK1 and IRF3 mRNAs were reduced in RIG-I-like receptor (RLR) pathway. In conclusion, BCoV-N reduced IFN-β levels in RIG-I-like receptor (RLR) pathway in HEK 293 T cells which were induced by MDA5, MAVS, TBK1 and IRF3(5D). BCoV-N protein inhibited IFN-β production and activation of RIG-I-like receptors (RLRs) signal pathway. Our findings demonstrated BCoV N protein is an IFN-β antagonist through inhibition of MDA5, MAVS, TBK1 and IRF3(5D) in RLRs pathway, also revealed a new mechanism of BCoV N protein to evade host innate immune response by inhibiting type I IFN production, which is beneficial to developing novel prevention strategy for BCoV disease in the animals and humans.

Transcriptome Analysis of Retinoic Acid-Inducible Gene I Overexpression Reveals the Potential Genes for Autophagy-Related Negative Regulation.

Retinoic acid-inducible gene I (RIG-I) serves as an essential viral RNA sensor for innate immune. The activation of the RIG-I-like receptors (RLRs) pathway triggers many regulations for the outcome of type I interferon, including ubiquitination, dephosphorylation, ISGylation, and autophagy. However, the autophagy-related regulation of RIG-I is still not fully understood. To investigate the potentially unknown genes related to autophagy-related regulation of RIG-I, we firstly confirm the induction of autophagy derived by overexpression of RIG-I. Furthermore, the autophagy inducer and inhibitor drugs were used in different assays. The results showed autophagy could control the activation of RLRs pathway and expression of exogenous RIG-I. In addition, we carried out the transcriptome analysis of overexpression of RIG-I in vitro. Differentially expressed genes (DEGs) in GO and KEGG signaling pathways enrichment provided a newly complex network. Finally, the validation of qPCR indicated that the DEGs PTPN22, PRKN, OTUD7B, and SIRT2 were correlated to the negative regulation of excessive expression of RIG-I. Taken together, our study contributed new insights into a more comprehensive understanding of the regulation of excessive expression of RIG-I. It provided the potential candidate genes for autophagy-related negative regulation for further investigation.

Transcriptional expression analysis reveals multiple effects of nonylphenol exposure on scallop immune system

Nonylphenol (NP) is an endocrine disruptor and environmental hormone representing alkylphenol compounds. Marine mollusks are an important source of protein for people worldwide. Many researchers have begun to study the effect of NP on marine mollusks immune system in view of its toxicity; however, the underlying molecular mechanisms require in-depth analysis. In this study, we focused on the transcriptional expression change of immune-related genes and antioxidant enzymes activities variation after NP exposure in a marine bivalve mollusk, Chlamys farreri, to explore the immunomodulatory capacity of NP in marine mollusks. We identified MAVS (Mitochondrial antiviral signaling protein), a key adaptor molecule in the RLR (RIG-I like receptor) pathway, and studied the expression of multiple immune-related genes in response to different concentrations of NP. The key genes involved in RLR/TLR (Toll like receptor) innate immune pathway, apoptosis, and cellular antioxidation mechanism were investigated. Changes in the enzymatic activities of scallop antioxidant enzymes after NP exposure were also examined. The results revealed that the genes expression and the antioxidant enzymes activities show significant changes, thus proving that NP stimulation affects the scallop immune system. Our research results demonstrate the immunomodulatory capacity of NP in marine bivalve mollusks and lay the foundation for further in-depth analysis of the molecular mechanism of NP toxicity.

In Vitro Characterization of the Innate Immune Pathways Engaged by Live and Inactivated Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) infection can lead to inflammation of the central nervous system. The disease can be effectively prevented by whole inactivated virus vaccines. Here, we investigated the innate immune profile induced in vitro by the antigen component of the vaccines, inactivated TBEV (I-TBEV), to gain insights into the mechanism of action of the TBE vaccine as compared to the live virus. To this end, we exposed human peripheral blood mononuclear cells (PBMCs) to inactivated and live TBEV and assessed cellular responses by RNA sequencing. Both inactivated and live TBEV significantly induced an interferon-dominated gene signature and an increased RIG-I-like receptor (RLR) expression. Using pathway-specific inhibitors, we assessed the involvement of pattern recognition receptors in the sensing of inactivated or live TBEV. Only RLR pathway inhibition significantly suppressed the downstream cascade induced by I-TBEV, while responses to the replicating virus were impacted by the inhibition of RIG-I-like, as well as Toll-like, receptors. Our results show that inactivated and live TBEV predominantly engaged an interferon response in our in vitro PBMC platform, and indicate RLRs as the main pattern recognition receptors involved in I-TBEV sensing.

Viperin interacts with PEX19 to mediate peroxisomal augmentation of the innate antiviral response.

Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-β. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

Targeting the RIG-I-like receptor signaling pathway to improve the efficacy of immunotherapy in mucosal and uveal melanoma.

e21593 Background: Patients with cutaneous (CM) or acral (AM) melanoma have high response rates to immune checkpoint blockade (ICB) therapies including anti-CTLA4, anti-PD1, and their combination. In contrast, patients with mucosal (MM) or uveal (UM) melanoma have low ICB response rates and a poorer overall prognosis. In this study, we analyzed melanoma transcriptomes to identify potential mechanisms underlying ICB resistance in MM and UM. We also evaluated epigenetic modifying drugs as potential agents to improve ICB responses in these patients. Methods: RNA sequencing was performed on 13 MM, 8 AM, and 24 CM patient-derived xenograft tumors. These data were analyzed along with published RNA sequencing data for 80 UM and 443 CM tumors. MM and UM cells were treated with several different classes of epigenetic modifying drugs, and the ICB-resistant YUMM1.7 mouse melanoma syngeneic model was used to evaluate the tumor growth effect of one particular drug, decitabine, at low (2.5 ug) and intermediate (10 ug) doses in combination with anti-PD1 (200 ug). Results: We identified 2513 differentially expressed genes (DEG) between MM and CM tumors (ANODEV adjusted p-value &lt; 0.05, FDR &lt; 0.1) compared to only 495 DEG between AM and CM. Ingenuity Pathway Analysis (IPA) analysis of the 2513 DEG in MM identified the RIG-I-like receptor (RLR) pathway as inhibited in MM versus CM ( p &lt; 1x10-5). The RLR pathway was also inhibited in ICB-resistant CM versus ICI-sensitive (anti-CTLA4, p &lt; 0.006 and anti-PD1, p &lt; 0.0002) and in UM versus CM ( p &lt; 1x10-8), but was not inhibited in AM. The RLR pathway is an innate immune pathogen sensing pathway that is critical for ICB responses in CM; therefore, we sought to identify FDA-approved drugs which activate the RLR pathway in MM and UM. Since RLR signaling is frequently repressed by epigenetic silencing, we screened direct DNMT1 inhibitors 5’-azacitidine (AZA) and 5’-aza-deoxycitidine (decitabine, DEC), several indirect DNMT1 inhibitors, and two HDAC inhibitors. We found that only DEC strongly induced the expression of RLR pathway genes in MM, UM, and ICB-resistant CM cells. Compared to vehicle, treatment of YUMM1.7 tumors with single agent anti-PD1 ( p &gt; 0.87) and low dose ( p &gt; 0.90) or intermediate dose ( p &gt; 0.80) DEC had no effect on tumor growth. However, the addition of either low or intermediate dose DEC to anti-PD1 showed a trend of decreased tumor growth (low dose, 31% decrease, p = 0.22 and intermediate dose, 41% decrease, p = 0.55). Conclusions: We identified low RLR pathway signaling as one possible mechanism underlying ICB resistance in MM and UM. We show that an FDA-approved DNMT1 inhibitor, decitabine, strongly induces RLR pathway genes in MM and UM cells in vitro and leads to tumor growth suppression in combination with anti-PD1 in vivo. Therefore, co-administration of decitabine and anti-PD1 agents is a rational and novel strategy for improving outcomes for MM and UM patients.

Viral Evasion of RIG-I-Like Receptor-Mediated Immunity through Dysregulation of Ubiquitination and ISGylation.

Viral dysregulation or suppression of innate immune responses is a key determinant of virus-induced pathogenesis. Important sensors for the detection of virus infection are the RIG-I-like receptors (RLRs), which, in turn, are antagonized by many RNA viruses and DNA viruses. Among the different escape strategies are viral mechanisms to dysregulate the post-translational modifications (PTMs) that play pivotal roles in RLR regulation. In this review, we present the current knowledge of immune evasion by viral pathogens that manipulate ubiquitin- or ISG15-dependent mechanisms of RLR activation. Key viral strategies to evade RLR signaling include direct targeting of ubiquitin E3 ligases, active deubiquitination using viral deubiquitinating enzymes (DUBs), and the upregulation of cellular DUBs that regulate RLR signaling. Additionally, we summarize emerging new evidence that shows that enzymes of certain coronaviruses such as SARS-CoV-2, the causative agent of the current COVID-19 pandemic, actively deISGylate key molecules in the RLR pathway to escape type I interferon (IFN)-mediated antiviral responses. Finally, we discuss the possibility of targeting virally-encoded proteins that manipulate ubiquitin- or ISG15-mediated innate immune responses for the development of new antivirals and vaccines.

Transcriptome analysis reveals temperature-dependent early immune response in flounder (Paralichthys olivaceus) after Hirame novirhabdovirus (HIRRV) infection

Hirame novirhabdovirus (HIRRV), as a highly pathogenic fish virus, is frequently prevalent in a variety of aquaculture fish in recent years, which seriously threatens the healthy development of aquaculture industry. Epidemiological studies show that the outbreak of HIRRV is obviously temperature dependent. Virus proliferation experiments in vitro and in vivo at different temperatures indicate the antiviral response of flounder is a main reason affect the replication of HIRRV. The RNA-Seq was used to analyze the different antiviral response in flounder which infected with HIRRV at different temperatures, the experiment set two temperatures of 10 °C and 20 °C. The flounder infected with HIRRV by artificial injection, and the spleens were collected at 24 h after infection. Meanwhile, the fish injected with EPC supernatant at different temperatures were set as control groups. It can obtain four pairwise comparison groups if determine a single variable, and the differentially expressed genes were further selected. The results showed that after infection with HIRRV at 10 °C and 20 °C, the differentially expressed genes in spleen were significantly enriched in inflammatory and immune-related pathways like Arachidonic acid metabolism, Cytokine-cytokine receptor interaction, Toll-like receptor (TLR) signaling pathway, RIG-I-like receptor (RLRs) signaling pathway, NOD-like receptor (NLR) signaling pathway and Cytosolic DNA-sensing pathway etc. In addition, the expression of phagocytes, lysosomes, endocytosis related genes were significantly upregulated at high temperature whether HIRRV positive or not. But compared to the infected flounder at 10 °C, some genes of RLRs signaling pathway were significantly upregulated at 20 °C, it can be speculated that RLRs pathway may be related to the anti-HIRRV response of flounder. Therefore, key genes of RLRs signaling pathway including mda5, lgp2, mita, mavs, irf3, irf7, ifn I-3 and ifn-γ were selected, and the temporal expression patterns of these genes in infected flounder at different temperatures were further detected by qRT-PCR. The results showed that HIRRV infection can significantly stimulate and activate the RLRs pathway of flounder, and the response level of this pathway was significantly higher at 20 °C than 10 °C. In general, this study provides important data for the further study about the pathogenesis of HIRRV infection in flounder.

RIG-I-like receptor activation drives type I IFN and antiviral signaling to limitHantaan orthohantavirus replication.

Pathogenic hantaviruses, genus Orthohantaviridae, are maintained in rodent reservoirs with zoonotic transmission to humans occurring through inhalation of rodent excreta. Hantavirus disease in humans is characterized by localized vascular leakage and elevated levels of circulating proinflammatory cytokines. Despite the constant potential for deadly zoonotic transmission to humans, specific virus-host interactions of hantaviruses that lead to innate immune activation, and how these processes impart disease, remain unclear. In this study, we examined the mechanisms of viral recognition and innate immune activation of Hantaan orthohantavirus (HTNV) infection. We identified the RIG-I-like receptor (RLR) pathway as essential for innate immune activation, interferon (IFN) production, and interferon stimulated gene (ISG) expression in response to HTNV infection in human endothelial cells, and in murine cells representative of a non-reservoir host. Our results demonstrate that innate immune activation and signaling through the RLR pathway depends on viral replication wherein the host response can significantly restrict replication in target cells in a manner dependent on the type 1 interferon receptor (IFNAR). Importantly, following HTNV infection of a non-reservoir host murine model, IFNAR-deficient mice had higher viral loads, increased persistence, and greater viral dissemination to lung, spleen, and kidney compared to wild-type animals. Surprisingly, this response was MAVS independent in vivo. Innate immune profiling in these tissues demonstrates that HTNV infection triggers expression of IFN-regulated cytokines early during infection. We conclude that the RLR pathway is essential for recognition of HTNV infection to direct innate immune activation and control of viral replication in vitro, and that additional virus sensing and innate immune response pathways of IFN and cytokine regulation contribute to control of HTNV in vivo. These results reveal a critical role for innate immune regulation in driving divergent outcomes of HTNV infection, and serve to inform studies to identify therapeutic targets to alleviate human hantavirus disease.

BICP0 Negatively Regulates TRAF6-Mediated NF-κB and Interferon Activation by Promoting K48-Linked Polyubiquitination of TRAF6.

The infected cell protein 0 (BICP0) is an immediate early protein encoded by BHV-1, and its RING finger domain, which endows BICP0 with intrinsic E3 ubiquitin ligase activity, is common in all ICP0 proteins. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the TRAF family members and is ubiquitously expressed in mammalian tissues. TRAF6 forms the MyD88-TRAF6-IRF7 complex and activates interferon induction in the TLR (Toll-like receptors) and the RLR (RIG-I-like receptor) pathway. Previous studies showed that BICP0 reduced IFN-β promoter activity by interacting with IRF7. In this study, we found that BICP0 promoted the K48-ubiquitination and degradation of TRAF6 through the ubiquitin proteasome system. The interaction between BICP0 and TRAF6 is a prerequisite for ubiquitination modification, and the 346-PAERQY-351 of BICP0 is indispensable. The motif mutation experiments showed that the tyrosine 351 of BICP0 is the key amino acid involved. Further studies demonstrated that BICP0 suppressed the NF-κB pathway via the interference of TRAF6. Moreover, degradation of TRAF6 protein influenced the K63-linked ubiquitination of IRF7 and activation of interferon promoter. Collectively, these findings indicate that the BICP0 protein suppresses the inflammation signaling and IFN production by K48-linked polyubiquitination of TRAF6 and may further clarify the immune evasion function of BICP0.

Cytoplasmic RNA Sensor Pathways and Nitazoxanide Broadly Inhibit Intracellular Mycobacterium tuberculosis Growth.

SummaryTo establish stable infection, Mycobacterium tuberculosis (MTb) must overcome host innate immune mechanisms, including those that sense pathogen-derived nucleic acids. Here, we show that the host cytosolic RNA sensing molecules RIG-I-like receptor (RLR) signaling proteins RIG-I and MDA5, their common adaptor protein MAVS, and the RNA-dependent kinase PKR each independently inhibit MTb growth in human cells. Furthermore, we show that MTb broadly stimulates RIG-I, MDA5, MAVS, and PKR gene expression and their biological activities. We also show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits intracellular MTb growth and amplifies MTb-stimulated RNA sensor gene expression and activity. This study establishes prototypic cytoplasmic RNA sensors as innate restriction factors for MTb growth in human cells and it shows that targeting this pathway is a potential host-directed approach to treat tuberculosis disease.

Peste des Petits Ruminants Virus Nucleocapsid Protein Inhibits Beta Interferon Production by Interacting with IRF3 To Block Its Activation.

Peste des petits ruminants virus (PPRV) is the etiological agent of peste des petits ruminants, causing acute immunosuppression in its natural hosts. However, the molecular mechanisms by which PPRV antagonizes the host immune responses have not been fully characterized. In particular, how PPRV suppresses the activation of the host RIG-I-like receptor (RLR) pathway has yet to be clarified. In this study, we demonstrated that PPRV infection significantly suppresses RLR pathway activation and type I interferon (IFN) production and identified PPRV N protein as an extremely important antagonistic viral factor that suppresses beta interferon (IFN-β) and IFN-stimulated gene (ISG) expression. A detailed analysis showed that PPRV N protein inhibited type I IFN production by targeting interferon regulatory factor 3 (IRF3), a key molecule in the RLR pathway required for type I IFN induction. PPRV N protein interacted with IRF3 (but not with other components of the RLR pathway, including MDA5, RIG-I, VISA, TBK1, and MITA) and abrogated the phosphorylation of IRF3. As expected, PPRV N protein also considerably impaired the nuclear translocation of IRF3. The TBK1-IRF3 interaction was involved significantly in IRF3 phosphorylation, and we showed that PPRV N protein inhibits the association between TBK1 and IRF3, which in turn inhibits IRF3 phosphorylation. The amino acid region 106 to 210 of PPRV N protein was determined to be essential for suppressing the nuclear translocation of IRF3 and IFN-β production, and the 140 to 400 region of IRF3 was identified as the crucial region for the N-IRF3 interaction. Together, our findings demonstrate a new mechanism evolved by PPRV to inhibit type I IFN production and provide structural insights into the immunosuppression caused by PPRV.IMPORTANCE Peste des petits ruminants is a highly contagious animal disease affecting small ruminants, which threatens both small livestock and endangered susceptible wildlife populations in many countries. The causative agent, peste des petits ruminants virus (PPRV), often causes acute immunosuppression in its natural hosts during infection. Here, for the first time, we demonstrate that N protein, the most abundant protein of PPRV, plays an extremely important role in suppression of interferon regulatory factor 3 (IRF3) function and type I interferon (IFN) production by interfering with the formation of the TBK1-IRF3 complex. This study explored a novel antagonistic mechanism of PPRV.

Alternative Splicing Transcripts of Zebrafish LGP2 Gene Differentially Contribute to IFN Antiviral Response.

In mammals, RIG-I like receptors (RLRs) RIG-I and melanoma differentiation-associated gene 5 (MDA5) sense cytosolic viral RNA, leading to IFN antiviral response; however, LGP2 exhibits controversial functions. The same happens to fish LGP2. In this study we report that three zebrafish LGP2 splicing transcripts, a full-length LGP2, and two truncating variants, LGP2v1 and LGP2v2, play distinct roles during IFN antiviral response. Overexpression of the full-length LGP2 not only potentiates IFN response through the RLR pathway, in the absence or presence of poly(I:C) at limited concentrations, but also inhibits IFN response by relative high concentrations of poly(I:C) through functional attenuation of signaling factors involved in the RLR pathway; however, LGP2v1 and LGP2v2 only retain the inhibitory role. Consistently, LGP2 but not LGP2v1 and LGP2v2 confers protection on fish cells against spring viremia of carp virus (SVCV) infection and at limited expression levels, LGP2 exerts more significant protection than either RIG-I or MDA5. Further data suggest that in the early phase of SVCV infection, LGP2 functions as a positive regulator but along with SVCV replicating in cells up to a certain titer, which leads to a far more robust expression of IFN, LGP2 switches to a negative role. These in vitro results suggest an ingenious mechanism where the three zebrafish LGP2 splicing transcripts work cooperatively to shape IFN antiviral responses.

Characterization of the Mollusc RIG-I/MAVS Pathway Reveals an Archaic Antiviral Signalling Framework in Invertebrates

Despite the mitochondrial antiviral signalling protein (MAVS)-dependent RIG-I-like receptor (RLR) signalling pathway in the cytosol plays an indispensable role in the antiviral immunity of the host, surprising little is known in invertebrates. Here we characterized the major members of RLR pathway and investigated their signal transduction a Molluscs. We show that genes involved in RLR pathway were significantly induced during virus challenge, including CgRIG-I-1, CgMAVS, CgTRAF6 (TNF receptor-associated factor 6), and CgIRFs (interferon regulatory factors. Similar to human RIG-I, oyster RIG-I-1 could bind poly(I:C) directly in vitro and interact with oyster MAVS via its caspase activation and recruitment domains. We also show that transmembrane domain-dependent self-association of CgMAVS may be crucial for its signalling and that CgMAVS can recruit the downstream signalling molecule, TRAF6, which can subsequently activate NF-κB signal pathway. Moreover, oyster IRFs appeared to function downstream of CgMAVS and were able to activate the interferon β promoter and interferon stimulated response elements in mammalian cells. These results establish invertebrate MAVS-dependent RLR signalling for the first time and would be helpful for deciphering the antiviral mechanisms of invertebrates and understanding the development of the vertebrate RLR network.