Structural analysis of RIG-I-like receptors reveals ancient rules of engagement between diverse RNA helicases and TRIM ubiquitin ligases.

RNA helicases and E3 ubiquitin ligases mediate many critical functions in cells, but their actions have largely been studied in distinct biological contexts. Here, we uncover evolutionarily conserved rules of engagement between RNA helicases and tripartite motif (TRIM) E3 ligases that lead to their functional coordination in vertebrate innate immunity. Using cryoelectron microscopy and biochemistry, we show that RIG-I-like receptors (RLRs), viral RNA receptors with helicase domains, interact with their cognate TRIM/TRIM-like E3 ligases through similar epitopes in the helicase domains. Their interactions are avidity driven, restricting the actions of TRIM/TRIM-like proteins and consequent immune activation to RLR multimers. Mass spectrometry and phylogeny-guided biochemical analyses further reveal that similar rules of engagement may apply to diverse RNA helicases and TRIM/TRIM-like proteins. Our analyses suggest not only conserved substrates for TRIM proteins but also, unexpectedly, deep evolutionary connections between TRIM proteins and RNA helicases, linking ubiquitin and RNA biology throughout animal evolution.

Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

Impaired interferon response in senecavirus A infection and identification of 3Cpro as an antagonist.

Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3Cpro) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3Cpro selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3Cpro cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3Cpro partially differ from previously reported classical motifs recognized by other picornaviral 3Cpro, highlighting the distinct characteristics of SVA 3Cpro. Together, these results reveal a mechanism by which SVA 3Cpro antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3Cpro.IMPORTANCESenecavirus A (SVA), the only member in the Senecavirus genus within the Picornaviridae family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3Cpro, a protease of SVA, functions as an antagonist for the IFN response. 3Cpro utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3Cpro as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.

Picornavirus infection enhances aspartate by the SLC38A8 transporter to promote viral replication.

Foot-and-mouth disease, a class of animal diseases, is caused by foot-and-mouth disease virus (FMDV). The metabolic changes during FMDV infection remain unclear. Here, PK-15 cells, serum, and tonsils infected with FMDV were analyzed by metabolomics. A total of 284 metabolites in cells were significantly changed after FMDV infection, and most of them belong to amino acids and nucleotides. Further studies showed that FMDV infection significantly enhanced aspartate in vitro and in vivo. The amino acid transporter solute carrier family 38 member 8 (SLC38A8) was responsible for FMDV-upregulated aspartate. Enterovirus 71 (EV71) and Seneca Valley virus (SVV) infection also enhanced aspartate by SLC38A8. Aspartate aminotransferase activity was also elevated in FMDV-, EV71-, and SVV-infected cells, which may lead to reversible transition between the TCA cycle and amino acids synthesis. Aspartate and SLC38A8 were essential for FMDV, EV71, and SVV replication in cells. In addition, aspartate and SLC38A8 also promoted FMDV and EV71 replication in mice. Detailed analysis indicated that FMDV infection promoted the transfer of mTOR to lysosome to enhance interaction between mTOR and Rheb, and activated PI3K/AKT/TSC2/Rheb/mTOR/p70S6K1 pathway to promote viral replication. The mTORC1 signaling pathway was responsible for FMDV-induced SLC38A8 protein expression. For the first time, our data identified metabolic changes during FMDV infection. These data identified a novel mechanism used by FMDV to upregulate aspartate to promote viral replication and will provide new perspectives for developing new preventive strategies.

NOG1 downregulates type I interferon production by targeting phosphorylated interferon regulatory factor 3.

The innate immune system is the first line of the host's defense, and studying the mechanisms of the negative regulation of interferon (IFN) signaling is important for maintaining the balance of innate immune responses. Here, we found that the host GTP-binding protein 4 (NOG1) is a negative regulator of innate immune responses. Overexpression of NOG1 inhibited viral RNA- and DNA-mediated signaling pathways, and NOG1 deficiency promoted the antiviral innate immune response, resulting in the ability of NOG1 to promote viral replication. Vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) infection induced a higher level of IFN-ß protein in NOG1 deficient mice. Meanwhile, NOG1-deficient mice were more resistant to VSV and HSV-1 infection. NOG1 inhibited type I IFN production by targeting IRF3. NOG1 was also found to interact with phosphorylated IFN regulatory factor 3 (IRF3) to impair its DNA binding activity, thereby downregulating the transcription of IFN-ß and downstream IFN-stimulated genes (ISGs). The GTP binding domain of NOG1 is responsible for this process. In conclusion, our study reveals an underlying mechanism of how NOG1 negatively regulates IFN-ß by targeting IRF3, which uncovers a novel role of NOG1 in host innate immunity.

Innate sensing of picornavirus infection involves cGAS-STING-mediated antiviral responses triggered by mitochondrial DNA release.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-ß production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.

Tyrosine phosphorylation of IRF3 by BLK facilitates its sufficient activation and innate antiviral response.

Viral infection triggers the activation of transcription factor IRF3, and its activity is precisely regulated for robust antiviral immune response and effective pathogen clearance. However, how full activation of IRF3 is achieved has not been well defined. Herein, we identified BLK as a key kinase that positively modulates IRF3-dependent signaling cascades and executes a pre-eminent antiviral effect. BLK deficiency attenuates RNA or DNA virus-induced ISRE activation, interferon production and the cellular antiviral response in human and murine cells, whereas overexpression of BLK has the opposite effects. BLK-deficient mice exhibit lower serum cytokine levels and higher lethality after VSV infection. Moreover, BLK deficiency impairs the secretion of downstream antiviral cytokines and promotes Senecavirus A (SVA) proliferation, thereby supporting SVA-induced oncolysis in an in vivo xenograft tumor model. Mechanistically, viral infection triggers BLK autophosphorylation at tyrosine 309. Subsequently, activated BLK directly binds and phosphorylates IRF3 at tyrosine 107, which further promotes TBK1-induced IRF3 S386 and S396 phosphorylation, facilitating sufficient IRF3 activation and downstream antiviral response. Collectively, our findings suggest that targeting BLK enhances viral clearance via specifically regulating IRF3 phosphorylation by a previously undefined mechanism.

African Swine Fever Virus E184L Protein Interacts with Innate Immune Adaptor STING to Block IFN Production for Viral Replication and Pathogenesis.

African swine fever is one of the most serious viral diseases that affects domestic and wild pigs. The causative agent, African swine fever virus (ASFV), has evolved sophisticated immune evasion mechanisms that target both innate and adaptive immune responses. However, the underlying molecular mechanisms have not been fully understood. Here, we report that ASFV E184L protein inhibits host innate immune response via targeting the stimulator of IFN genes (STING)-mediated signaling pathway in both human embryonic kidney HEK-293T cells and porcine pulmonary alveolar macrophages. E184L interacts with STING, impairing dimerization and oligomerization of STING but not affecting its puncta formation at the perinuclear region. Furthermore, E184L disrupts STING-TBK1-IRF3 complex formation, leading to inhibition of STING phosphorylation, and IRF3 dimerization and nuclear translocation. The 1-20 aa region in E184L is essential for E184L-STING interaction and blocking IL-1ß and type I IFN production. Deletion of E184L in ASFV considerably impairs antagonistic function of the virus in suppression of the STING-mediated antiviral response, an effect that is reversible by introduction of E184L. Importantly, the virulence of mutant ASFV lacking E184L is reduced in pigs compared with its parental virus due to induction of higher IFN production in vivo. Our findings indicate that ASFV E184L is an important antagonist of IFN signaling to evade host innate immune antiviral responses, which improves our understanding of immune evasion mechanisms of ASFV.

From homeostasis to defense: Exploring the role of selective autophagy in innate immunity and viral infections.

The process of autophagy, a conservative evolutionary mechanism, is responsible for the removal of surplus and undesirable cytoplasmic components, thereby ensuring cellular homeostasis. Autophagy exhibits a remarkable level of selectivity by employing a multitude of cargo receptors that possess the ability to bind both ubiquitinated cargoes and autophagosomes. In the context of viral infections, selective autophagy plays a crucial role in regulating the innate immune system. Notably, numerous viruses have developed strategies to counteract, evade, or exploit the antiviral effects of selective autophagy. This review encompasses the latest research progress of selective autophagy in regulating innate immunity and virus infectious.

Pt-Cluster-Embedded Metal-Organic Frameworks-Derived Fe@C as Dual-Enzyme Mimics for NADH Detection in Serum.

Dihydro-nicotinamide adenine dinucleotide (NADH) detection is crucial since it is a vital coenzyme in organism metabolism. Compared to the traditional method based on natural NADH oxidase (NOX), nanozymes with multienzyme-like activity can catalyze multistage reactions in a singular setup, simplifying detection processes and enhancing sensitivity. In this study, an innovative NADH detection method was developed using iron-doped carbon (Fe@C) nanozyme synthesized from metal-organic frameworks with in situ reduced Pt clusters. This nanozyme composite (Pt/Fe@C) demonstrated dual NOX and peroxidase-like characteristics, significantly enhancing the catalytic efficiency and enabling NADH conversion to NAD+ and H2O2 with subsequent detection. The collaborative research involving both experimental and theoretical simulations has uncovered the catalytic process and the cooperative effect of Fe and Pt atoms, leading to enhanced oxygen adsorption and activation, as well as a decrease in the energy barrier of the key step in the H2O2 decomposition process. These findings indicate that the catalytic performance of Pt/Fe@C in NOX-like and POD-like reactions can be significantly improved. The colorimetric sensor detects NADH with a limit of detection as low as 0.4 nM, signifying a breakthrough in enzyme-mimicking nanozyme technology for precise NADH measurement.

Peste Des Petits Ruminants Virus Nucleocapsid Protein Interacts with Protein Kinase R-Activating Protein and Induces Stress Granules To Promote Viral Replication.

The pathogenic mechanisms of peste des petits ruminants virus (PPRV) infection remain poorly understood, leaving peste des petits ruminants (PPR) control and eradication especially difficult. Here, we determined that PPRV nucleocapsid (N) protein triggers formation of stress granules (SGs) to benefit viral replication. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N protein interacted with protein kinase R (PKR)-activating protein (PACT), and this interaction was confirmed in the context of PPRV infection. PACT was essential for PPRV replication. Besides, the ectopic expression of N activated the PKR/eIF2α (α subunit of eukaryotic initiation factor 2) pathway through induction of PKR phosphorylation, but it did not induce PKR phosphorylation in PACT-deficient (PACT-/-) cells. PPRV N interacted with PACT, impairing the interaction between PACT and a PKR inhibitor, transactivation response RNA-binding protein (TRBP), which subsequently enhanced the interaction between PACT and PKR and thus promoted the activation of PKR and eIF2α phosphorylation, resulting in formation of stress granules (SGs). Consistently, PPRV infection induced SG formation through activation of the PKR/eIF2α pathway, and knockdown of N impaired PPRV-induced SG formation. PPRV-induced SG formation significantly decreased in PACT-/- cells as well. The role of SG formation in PPRV replication was subsequently investigated, which showed that SG formation plays a positive role in PPRV replication. By using an RNA fluorescence in situ hybridization assay, we found that PPRV-induced SGs hid cellular mRNA rather than viral mRNA. Altogether, our data provide the first evidence that PPRV N protein plays a role in modulating the PKR/eIF2α/SG axis and promotes virus replication through targeting PACT. IMPORTANCE Stress granule (SG) formation is a conserved cellular strategy to reduce stress-related damage regulating cell survival. A mass spectrometry-based profiling of the interactome of PPRV N protein revealed that PPRV N interacted with PACT to regulate the assembly of SGs. N protein inhibited the interaction between PACT and a PKR inhibitor, TRBP, through binding to the M1 domain of PACT, which enhanced the interaction between PACT and PKR and thus promoted PKR activation and subsequent eIF2α phosphorylation as well as SG formation. The regulatory function of N protein was strikingly abrogated in PACT-/- cells. SGs induced by PPRV infection through the PKR/eIF2α pathway are PACT dependent. The loss-of-function assay indicated that PPRV-induced SGs were critical for PPRV replication. We concluded that the PPRV N protein manipulates the host PKR/eIF2α/SG axis to favor virus replication.

Metabolomic analysis of pig spleen reveals African swine fever virus infection increased acylcarnitine levels to facilitate viral replication.

African swine fever (ASF) is a devastating disease caused by the African swine fever virus (ASFV) that adversely affects the pig industry. The spleen is the main target organ of ASFV; however, the function of metabolites in the spleen during ASFV infection is yet to be investigated. To define the metabolic changes in the spleen after ASFV infection, untargeted and targeted metabolomics analyses of spleens from ASFV-infected pigs were conducted. Untargeted metabolomics analysis revealed 540 metabolites with significant differential levels. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that these metabolites were mainly enriched in metabolic pathways, including nucleotide metabolism, purine metabolism, arginine biosynthesis, and neuroactive ligand-receptor interaction. Moreover, 134 of 540 metabolites quantified by targeted metabolomics analysis had differential levels and were enriched in metabolic pathways such as the biosynthesis of cofactors, ABC transporters, and biosynthesis of amino acids. Furthermore, coalition analysis of untargeted and targeted metabolomics data revealed that the levels of acylcarnitines, which are intermediates of fatty acid ß-oxidation, were significantly increased in ASFV-infected spleens compared with those in the uninfected spleens. Moreover, inhibiting fatty acid ß-oxidation significantly reduced ASFV replication, indicating that fatty acid ß-oxidation is essential for this process. To our knowledge, this is the first report presenting the metabolite profiles of ASFV-infected pigs. This study revealed a new mechanism of ASFV-mediated regulation of host metabolism. These findings provide new insights into the pathogenic mechanisms of ASFV, which will benefit the development of target drugs for ASFV replication. IMPORTANCE African swine fever virus, the only member of the Asfarviridae family, relies on hijacking host metabolism to meet the demand for self-replication. However, the change in host metabolism after African swine fever virus (ASFV) infection remains unknown. Here, we analyzed the metabolic changes in the pig spleen after ASFV infection for the first time. ASFV infection increased the levels of acylcarnitines. Inhibition of the production and metabolism of acylcarnitines inhibited ASFV replication. Acylcarnitines are the vital intermediates of fatty acid ß-oxidation. This study highlights the critical role of fatty acid ß-oxidation in ASFV infection, which may help identify target drugs to control African swine fever disease.

Deletion of DP148R, DP71L, and DP96R Attenuates African Swine Fever Virus, and the Mutant Strain Confers Complete Protection against Homologous Challenges in Pigs.

The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.

New role of gramicidin A in RIG-I-like receptors-mediated IFN signalling.

The pattern recognition receptors (PRRs) sense exogenous molecular patterns most commonly derived from invading pathogens, to active the interferon (IFN) signalling. In the cytoplasm, the viral double-stranded RNAs (dsRNAs) are sensed by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), depending on the length and chemical properties. Through the binding and oligomerizing onto the RNAs, they form filament to initiate the signalling cascade. Regulation of these receptors' activities are essential for manipulating the strength of IFN signalling. Here, through the virtual screening of chemical reagents using the published MDA5-dsRNA complex structure (PDB: 4GL2), we identified an antibiotic, gramicidin A as a stimulator that enhanced MDA5-mediated IFN signalling. Cytotoxic assay and IFN signalling assay suggested that disruption of lipid membrane, which is a well-defined mechanism of gramicidin A to perform its action, was dispensable in this process. Sucrose gradient ultracentrifugation assay showed that the gramicidin A treatment enhanced MDA5 oligomerization status in the presence of dsRNA. Our work implicated a new role of gramicidin A in innate immunity and presented a new tool to manipulate MDA5 activity.

African Swine Fever Virus Regulates Host Energy and Amino Acid Metabolism To Promote Viral Replication.

African swine fever is one of the most serious viral diseases caused by African swine fever virus (ASFV). The metabolic changes induced by ASFV infection remain unknown. Here, porcine alveolar macrophages (PAMs) infected with ASFV was analyzed by ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 90 metabolites were significantly changed after ASFV infection, and most of them were amino acids and tricarboxylic acid (TCA) cycle intermediates. ASFV infection induced an increase in most of amino acids in the host during the early stages of infection, and amino acids decreased in the late stages of infection. ASFV infection did not significantly affect the glycolysis pathway, whereas it induced increases in citrate, succinate, α-ketoglutarate, and oxaloacetate levels in the TCA cycle, suggesting that ASFV infection promoted the TCA cycle. The activities of aspartate aminotransferase and glutamate production were significantly elevated in ASFV-infected cells and pigs, resulting in reversible transition between TCA cycle and amino acid synthesis. Aspartate, glutamate, and TCA cycle were essential for ASFV replication. In addition, ASFV infection induced an increase in lactate level using lactate dehydrogenase, which led to low expression of beta interferon (IFN-ß) and increased ASFV replication. Our data, for the first time, indicate that ASFV infection controls IFN-ß production through RIG-I-mediated signaling pathways. These data identified a novel mechanism evolved by ASFV to inhibit host innate immune responses and provide insights for development of new preventive or therapeutic strategies targeting the altered metabolic pathways. IMPORTANCE In order to promote viral replication, viruses often cause severe immunosuppression and seize organelles to synthesize a large number of metabolites required for self-replication. African swine fever virus (ASFV) has developed many strategies to evade host innate immune responses. However, the impact of ASFV infection on host cellular metabolism remains unknown. Here, for the first time, we analyzed the metabolomic profiles of ASFV-infected PAMs. ASFV infection increased host TCA cycle and amino acid metabolism. Aspartate, glutamate, and TCA cycle promoted ASFV replication. ASFV infection also induced the increase of lactate production to inhibit innate immune responses for self-replication. This study identified novel immune evasion mechanisms utilized by ASFV and provided insights into ASFV-host interactions, which is critical for guiding the design of new prevention strategies against ASFV targeting the altered metabolic pathways.

FMDV 3A Antagonizes the Effect of ANXA1 to Positively Modulate Viral Replication.

The RIG-I-like receptor signaling pathway is crucial for producing type I interferon (IFN-I) against RNA viruses. The present study observed that viral infection increased annexin-A1 (ANXA1) expression, and ANXA1 then promoted RNA virus-induced IFN-I production. Compared to ANXA1 wild-type cells, ANXA1-/- knockout cells showed IFN-ß production decreasing after viral stimulation. RNA virus stimulation induced ANXA1 to regulate IFN-ß production through the TBK1-IRF3 axis but not through the NF-κB axis. ANXA1 also interacted with JAK1 and STAT1 to increase signal transduction induced by IFN-ß or IFN-γ. We assessed the effect of ANXA1 on the replication of foot-and-mouth disease virus (FMDV) and found that ANXA1 inhibits FMDV replication dependent on IFN-I production. FMDV 3A plays critical roles in viral replication and host range. The results showed that FMDV 3A interacts with ANXA1 to inhibit its ability to promote IFN-ß production. We also demonstrated that FMDV 3A inhibits the formation of ANXA1-TBK1 complex. These results indicate that ANXA1 positively regulates RNA virus-stimulated IFN-ß production and FMDV 3A antagonizes ANXA1-promoted IFN-ß production to modulate viral replication. IMPORTANCE FMDV is a pathogen that causes one of the world's most destructive and highly contagious animal diseases. The FMDV 3A protein plays a critical role in viral replication and host range. Although 3A is one of the viral proteins that influences FMDV virulence, its underlying mechanisms remain unclear. ANXA1 is involved in immune activation against pathogens. The present study demonstrated that FMDV increases ANXA1 expression, while ANXA1 inhibits FMDV replication. The results also showed that ANXA1 promotes RNA virus-induced IFN-I production through the IRF3 axis at VISA and TBK1 levels. ANXA1 was also found to interact with JAK1 and STAT1 to strengthen signal transduction induced by IFN-ß and IFN-γ. 3A interacted with ANXA1 to inhibit ANXA1-TBK1 complex formation, thereby antagonizing the inhibitory effect of ANXA1 on FMDV replication. This study helps to elucidate the mechanism underlying the effect of the 3A protein on FMDV replication.

JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting RNF5 to promote activated IRF3 K48 ubiquitination.

The negative regulation of antiviral immune responses is essential for the host to maintain homeostasis. Jumonji domain-containing protein 6 (JMJD6) was previously identified with a number of functions during RNA virus infection. Upon viral RNA recognition, retinoic acid-inducible gene-I-like receptors (RLRs) physically interact with the mitochondrial antiviral signaling protein (MAVS) and activate TANK-binding kinase 1 (TBK1) to induce type-I interferon (IFN-I) production. Here, JMJD6 was demonstrated to reduce type-I interferon (IFN-I) production in response to cytosolic poly (I:C) and RNA virus infections, including Sendai virus (SeV) and Vesicular stomatitis virus (VSV). Genetic inactivation of JMJD6 enhanced IFN-I production and impaired viral replication. Our unbiased proteomic screen demonstrated JMJD6 contributes to IRF3 K48 ubiquitination degradation in an RNF5-dependent manner. Mice with gene deletion of JMJD6 through piggyBac transposon-mediated gene transfer showed increased VSV-triggered IFN-I production and reduced susceptibility to the virus. These findings classify JMJD6 as a negative regulator of the host's innate immune responses to cytosolic viral RNA.

Proteins in pregnant swine serum promote the African swine fever virus replication: an iTRAQ-based quantitative proteomic analysis.

African swine fever (ASF) is a severe infectious disease caused by the African swine fever virus (ASFV), seriously endangering the global pig industry. ASFV possesses a large genome, strong mutation ability, and complex immune escape mechanisms. Since the first case of ASF was reported in China in August 2018, it has had a significant impact on social economy and food safety. In the present study, pregnant swine serum (PSS) was found to promote viral replication; differentially expressed proteins (DEPs) in PSS were screened and identified using the isobaric tags for relative and absolute quantitation technology and compared with those in non-pregnant swine serum (NPSS). The DEPs were analyzed using Gene Ontology functional annotation, Kyoto Protocol Encyclopedia of Genes and Genome pathway enrichment, and protein-protein interaction networks. In addition, the DEPs were validated via western blot and RT-qPCR experiments. And the 342 of DEPs were identified in bone marrow-derived macrophages cultured with PSS compared with the NPSS. The 256 were upregulated and 86 of DEPs were downregulated. The primary biological functions of these DEPs involved signaling pathways that regulate cellular immune responses, growth cycles, and metabolism-related pathways. An overexpression experiment showed that the PCNA could promote ASFV replication whereas MASP1 and BST2 could inhibit it. These results further indicated that some protein molecules in PSS were involved in the regulation of ASFV replication. In the present study, the role of PSS in ASFV replication was analyzed using proteomics, and the study will be provided a basis for future detailed research on the pathogenic mechanism and host interactions of ASFV as well as new insights for the development of small-molecule compounds to inhibit ASFV.

Structural diversity and biological role of the 5' untranslated regions of picornavirus.

The genomic arrangement of most picornavirus of the Picornaviridae family shares a similar monocistronic genomic pattern and a defining organizational feature. A defining feature of picornavirus is the presence of evolutionarily conserved and highly-structured RNA elements in untranslated regions (UTRs) at the genome' 5'and 3' ends, essential for viral replication and translation. Given the diversity and complexity of RNA structure and the limitations of molecular biology techniques, the functional characterization and biological significance of UTRs remain to be fully elucidated, especially for 5' UTR. Here, we summarize the current knowledge of the 5' UTR of picornavirus. This review focuses on the structural characterization and the biological function of the RNA secondary and tertiary structures in the 5' UTR of picornavirus. Understanding the role of the 5' UTR of picornavirus can provide a deep insight into the viral replication cycle and pathogenic mechanisms.

Dysregulation of the RIG-I-like Receptor Pathway Signaling by Peste des Petits Ruminants Virus Phosphoprotein.

Peste des petits ruminants virus (PPRV) is a Morbillivirus that causes highly contagious and severe disease in various ruminants. PPRV infection leads to a severe inhibition of host antiviral immune response. Our previous study demonstrated that PPRV V protein blocks IFN response by targeting STAT proteins. In the current study, we identified the phosphoprotein (P) as a novel antagonistic factor of PPRV to counteract host antiviral innate immune response. PPRV P protein significantly suppressed RIG-I-like receptor pathway signaling and impaired IFN-ß and ISGs expression by targeting IFN regulatory factor (IRF)3 in both human embryonic kidney 293T cells and primary goat fibroblasts. The 1-102 region of P protein was critical for the antagonistic function of P protein. P protein interacted with IRF association domain (IAD) of IRF3 to block the interaction between TBK1 and IRF3. The interaction between TBK1 and the IAD of IRF3 is responsible for triggering the phosphorylation of IRF3. P protein competed with TBK1 to bind to the IAD of IRF3 that contributed to the decreased phosphorylation of IRF3, which, in turn, interfered with the dimerization of IRF3 and blocked IRF3 nuclear transportation. Besides, we also found that P protein interacted with IRF5 and IRF8. However, the involved mechanism remains unknown. Taken together, our results reveal a novel mechanism by which PPRV P protein antagonizes host antiviral innate immune response by interacting with the transcription factor IRF3, thereby inhibiting the type I IFN production and promoting viral replication.