Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

KIF5B-mediated internalization of FMDV promotes virus infection.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease, which is caused by the FMD virus (FMDV). Although the cell receptor for FMDV has been identified, the specific mechanism of FMDV internalization after infection remains unknown. In this study, we found that kinesin family member 5B (KIF5B) plays a vital role during FMDV internalization. Moreover, we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation (Co-IP) and co-localization in FMDV-infected cells. In particular, the stalk [amino acids (aa) 413-678] domain of KIF5B was indispensable for KIF5B-VP1 interaction. Moreover, overexpression of KIF5B dramatically enhanced FMDV replication; consistently, knockdown or knockout of KIF5B suppressed FMDV replication. Furthermore, we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating. KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection. In conclusion, our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport. This study may provide a new therapeutic target for developing FMDV antiviral drugs.

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.

Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication.

Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host's antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-ß promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection.Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-ß:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2.

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.

BLK positively regulates TLR/IL-1R signaling by catalyzing TOLLIP phosphorylation.

TLR/IL-1R signaling plays a critical role in sensing various harmful foreign pathogens and mounting efficient innate and adaptive immune responses, and it is tightly controlled by intracellular regulators at multiple levels. In particular, TOLLIP forms a constitutive complex with IRAK1 and sequesters it in the cytosol to maintain the kinase in an inactive conformation under unstimulated conditions. However, the underlying mechanisms by which IRAK1 dissociates from TOLLIP to activate TLR/IL-1R signaling remain obscure. Herein, we show that BLK positively regulates TLR/IL-1R-mediated inflammatory response. BLK-deficient mice produce less inflammatory cytokines and are more resistant to death upon IL-1ß challenge. Mechanistically, BLK is preassociated with IL1R1 and IL1RAcP in resting cells. IL-1ß stimulation induces heterodimerization of IL1R1 and IL1RAcP, which further triggers BLK autophosphorylation at Y309. Activated BLK directly phosphorylates TOLLIP at Y76/86/152 and further promotes TOLLIP dissociation from IRAK1, thereby facilitating TLR/IL-1R-mediated signal transduction. Overall, these findings highlight the importance of BLK as an active regulatory component in TLR/IL-1R signaling.

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.

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.

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.

African swine fever virus MGF360-9L promotes viral replication by degrading the host protein HAX1.

African swine fever virus (ASFV) infection causes African swine fever (ASF), a virulent infectious disease that threatens the safety of livestock worldwide. Studies have shown that MGF360-9 L is important for the virulence of ASFV and the host protein HS1-associated protein X-1 (HAX1) plays an important role in viral pathogenesis. This study aimed to clarify the mechanism by which HAX1 mediates ASFV replication through interactions with MGF360-9 L. The regions of interaction between MGF360-9 L and HAX1 were predicted and validated. HAX1 overexpression and RNA interference studies revealed that HAX1 is a host restriction factor that suppresses ASFV replication. Moreover, HAX1 expression was inhibited in ASFV-infected mature bone marrow-derived macrophages, and infection with the virulent MGF360-9 L gene deletion strain (∆MGF360-9 L) attenuated the inhibitory effect of the wild-type strain (WT) on HAX1 expression, suggesting a complex regulatory relationship between MGF360-9 L and HAX1. Furthermore, the E3 ubiquitin ligase RNF114 interacted with MGF360-9 L and HAX1, MGF360-9 L degraded HAX1 through the ubiquitin-proteasome pathway, and RNF114 facilitated the degradation of HAX1 by MGF360-9L-linked K48 ubiquitin chains through the ubiquitin-proteasome pathway, thereby facilitating ASFV replication. In conclusion, this study has enriched our understanding of the regulatory networks between ASFV proteins and host proteins and provided a reference for investigation into the pathogenesis and immune escape mechanism of ASFV.

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.

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.

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.

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.

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.

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.

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 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.

The endocytosis of foot-and mouth disease virus requires clathrin and caveolin and is dependent on the existence of Rab5 and Rab7 in CHO-677 cells.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes severe vesicular disease of cloven-hoofed animals. Various endocytosis mechanisms are involved in the entry of FMDV after binding to the integrin and heparan sulfate (HS) receptors. However, the mechanism of FMDV using other unknown receptors to enter the cells remains unclear. Here, we reported that the endocytosis and endosomal pathways are employed by FMDV to invade the Chinese hamster ovary cell line (CHO-677) without the integrin and HS receptors. We demonstrated that the internalization of FMDV into CHO-677 cells was abrogated by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. Knockdown of the clathrin heavy chain decreased the viral protein abundance. Incubation of the CHO-677 cells with the inhibitors of caveolae-mediated endocytosis or transfection by caveolin-1 siRNA also limited FMDV replication. In addition, we determined that the acidic environment and the existence of dynamin were essential for FMDV infection in CHO-677 cells. The endosomal proteins Rab5 (early endosome) and Rab7 (late endosome), but not Rab11 (recycling endosome), were utilized by FMDV during infection. These data provide a new entry model of FMDV by unknown receptors which will help to better understand the pathogenesis mediated by FMDV.