Lumpy skin disease (LSD) is an emerging transboundary viral disease of livestock caused by the lumpy skin disease virus (LSDV), which has caused substantial economic losses and negatively impacted veterinary public health. The underlying pathogenic mechanisms remain poorly understood, limiting the effective prevention and control of LSD. Here, we identify LSDV-encoded protein LSDV001 as a positive regulator of IL-1β- and TNFα-triggered signaling. LSDV001 interacts with TAK1 and TAB2/3 and promotes assembly of the TAK1-TAB2/3 complex. This leads to IKK-dependent activation of the transcription factor NF-κB and induction of downstream inflammatory cytokines. LSDV001-deficient virus (LSDVΔ001) has attenuated the ability to activate NF-κB and induce the expression of inflammatory cytokines. Infection with LSDVΔ001 leads to smaller skin nodules and reduced inflammation compared to wild-type LSDV. Our findings suggest that LSDV001 acts as a key virulence factor of LSDV by promoting excessive inflammatory response upon infection. IMPORTANCE Lumpy skin disease is a current global concern caused by the lumpy skin disease virus (LSDV), for which there is a lack of safe and efficient vaccines. In this study, we report that LSDV001 protein potentiates IL-1β- and TNFα-triggered IKK-dependent activation of NF-κB and transcription of inflammatory cytokines. Mechanistically, LSDV001 enhances inflammatory response by interacting with TAK1 and TAB2/3 to promote TAK1-TAB2/3 complex formation. We further demonstrate that LSDV001 deficiency attenuates LSDV-triggered inflammatory response and pathogenesis. Our findings identify a new virulence factor and reveal a novel pathogenic mechanism of LSDV by which LSDV001 enhances inflammatory response.
Opportunistic pathogens can cause infections when host defenses are compromised. Among them, Streptococcus suis (S. suis) colonizes the upper respiratory tract of pigs and causes severe diseases in both swine and humans. Although the pathogenic mechanisms of these bacteria have been partially elucidated, the molecular processes that govern their adaptation, colonization, and pathogenesis remain incompletely understood. In this study, we identified PrlP as a transcriptional repressor in S. suis that responds to mildly acidic, oxidative, hyperosmotic, and thermal stresses, and regulates bacterial growth, chain morphology, nasal colonization, and virulence. The C-terminal S24 peptidase domain of PrlP mediates stress-induced self-cleavage to control protein stability, while the N-terminal helix-turn-helix (HTH) DNA-binding domain is essential for its transcriptional regulatory function. Combined ChIP-seq and RNA-seq analyses revealed its binding motif (5'-CCTGAAWCT-3') and identified B9H01_08740 as a direct target gene, as further validated by EMSA. Notably, deletion of B9H01_08740 in the prlP-deficient background restored the associated phenotypes. These findings highlight PrlP as a key regulator in S. suis that maintains cellular homeostasis in response to stress conditions and modulates target genes such as B9H01_08740 to promote nasal colonization and virulence. Therefore, this study provides new insights into the regulatory mechanisms of pathogenic bacteria and may aid in the development of targeted strategies against S. suis infections.
The molecular mechanisms underlying the transport of influenza A virus (IAV) membrane proteins to the cell surface remain largely unclear. In this study, siRNA screening identifies Rab27a as a critical host factor regulating this transport process. GTP-bound Rab27a operates via its effectors, synaptotagmin-like protein 1 (SYTL1) and SYTL4, to facilitate the transport of vesicles carrying viral membrane proteins to the plasma membrane. Absence of Rab27a or SYTL4 does not block the early stages of the IAV life cycle but restricts viral assembly and budding. Notably, silencing SYTL4 provides superior protection in the female mouse IAV infection model. This investigation elucidates the molecular mechanism by which Rab27a and its effectors modulate the transport of IAV membrane proteins, thereby bridging a critical gap in IAV life cycle research and presenting a potential target for the development of antiviral drugs.
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a novel coronavirus that causes acute diarrhea, vomiting, and high mortality in suckling piglets. Research has demonstrated that certain viruses enhance their replication by modulating intracellular cholesterol metabolism. However, the impact of SADS-CoV infection on cellular cholesterol synthesis remains unclear. Here, we found that SADS-CoV Spike (S) protein promoted syncytium formation by positively regulating cholesterol synthesis. Specifically, the virus upregulated the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase through the inhibition of AMP-activated protein kinase (AMPK) activity. This inhibition was mediated by the activation of AKT-dependent phosphorylation of AMPKα at Ser485. Further investigation revealed that SADS-CoV S protein activated the PI3K/AKT pathway to promote cholesterol synthesis, a process that required the membrane protein integrin β1 (ITGB1). Importantly, we discovered that cholesterol facilitated cell-to-cell fusion mediated by the viral S protein, which enhanced syncytium formation. In summary, our findings demonstrate that the SADS-CoV S protein enhances cellular cholesterol accumulation by activating the PI3K/AKT/AMPK pathway through ITGB1, and that cholesterol facilitates syncytium formation mediated by the viral S protein. These insights contribute to a better understanding of SADS-CoV infection mechanisms and may inform future therapeutic strategies. IMPORTANCE Cholesterol, a vital component of cellular membranes, is crucial for maintaining cell structure and function. It also acts as an essential host factor for the entry, replication, and propagation of various viruses. In this study, we show that the Spike protein of swine acute diarrhea syndrome coronavirus (SADS-CoV) promotes syncytial formation by upregulating cellular cholesterol synthesis. The viral Spike protein activates the PI3K/AKT signaling pathway, leading to increased cholesterol production through the inhibition of AMP-activated protein kinase (AMPK). This upregulation of cholesterol facilitates cell-to-cell fusion, a process that enhances viral spread and pathogenesis. Moreover, we demonstrate that integrin β1 (ITGB1) acts as a critical host factor that links the viral Spike protein to the activation of the PI3K/AKT pathway. ITGB1 interacts with the S protein, playing a pivotal role in viral replication and cholesterol synthesis regulation. Our findings highlight the critical role of cholesterol in SADS-CoV infection and provide a deeper understanding of the molecular mechanisms behind viral replication. This research opens up potential therapeutic strategies targeting cholesterol metabolism to mitigate the effects of SADS-CoV and similar viral infections.
Classical swine fever virus (CSFV) infection induces complete mitophagy, which is essential for the clearance of damaged mitochondria. The endosomal sorting complex required for transport (ESCRT) machinery plays a vital role in mediating phagophore closure and autophagosome-lysosome fusion during starvation-induced autophagy. Nevertheless, its involvement in CSFV-induced mitophagy and the underlying mechanisms remain insufficiently understood. Here, we found that the ESCRT-III subunits including CHMP1A, CHMP1B, and CHMP4B, along with the AAA-ATPase VPS4, were actively recruited to autophagosomes during CSFV-induced mitophagy. Consistent with this, depletion of CHMP1A, CHMP1B, CHMP4B or VPS4A disrupted mitophagic flux, impairing both PINK1-PRKN-dependent and -independent pathways. Further investigations revealed that CSFV transiently recruited these subunits to nascent autophagosomes for phagophore sealing during mitophagy. Remarkably, multiple CSFV nonstructural proteins (NSPs) including NS3, NS4B, NS5A and NS5B interacted with these ESCRT key subunits and colocalized on mitophagosomes. Taken together, our study identifies CHMP1A, CHMP1B, CHMP4B, and VPS4A as pivotal regulators of phagophore closure in CSFV-induced mitophagy, unveiling novel mechanisms by which the virus manipulates host cellular pathways and highlighting potential therapeutic targets for infection control.Abbreviation: ATF4: activating transcription factor 4; ATG5: autophagy related 5; BafA1: bafilomycin A1; BFP: blue fluorescent protein; BNIP3L/NIX: BCL2 interacting protein 3like; BSA: bovine serum albumin; CALCOCO2/NDP52: calcium binding andcoiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHMP: charged multivesicular body protein; COX4: cytochrome c oxidase subunit 4; CSFV: classical swine fever virus; DAPI: 4',6-diamidino-2-phenylindole; DN: dominant-negative; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; hpt: hours post-transfection; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; IB: immunoblotting; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MFN2: mitofusin 2; MITO: mitochondria; MOI: multiplicity of infection; mtDNA: mitochondrial DNA; OPTN: optineurin; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAPA: rapamycin; RFP: redfluorescent protein; RT-qPCR: reverse transcription-quantitativereal-time polymerase chain reaction; RT-PCR: real-time polymerasechain reaction; SD: standard deviation; siCtrl: negative control siRNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1: voltage dependent anion channel 1; VPS4A: vacuolar protein sorting 4 homolog A; WCL: whole-cell lysate; WT: wild-type.
Macroautophagy/autophagy is a biological process that sequesters and degrades cytoplasmic material, damaged organelles, and infectious pathogens in eukaryotic cells via lysosomes. Autophagy is involved in different phases of the viral life cycle and regulates viral replication. Here, we demonstrated that pseudorabies virus (PRV) infection induced incomplete autophagy, and blocking the autophagosome-lysosome fusion facilitated PRV replication. Mechanistically, PRV late envelope glycoprotein M (gM) triggered SQSTM1/p62-dependent selective autophagy. Meanwhile, gM protein was found to inhibit the fusion between autophagosomes and lysosomes by activating CASP3 (caspase 3) to degrade SNAP29, resulting in increased viral replication. Interestingly, we confirmed that the gM homologs from several herpesviruses (herpes simplex virus-1, human cytomegalovirus, equine herpesvirus-1, and varicella-zoster virus) shared the same function of activating CASP3 and inhibiting autophagic flux. Deletion of the CASP3 gene led to an intact autophagic pathway and the increased formation of autolysosomes. Collectively, our results illustrated that blockage of autophagosome-lysosome fusion mediated by PRV gM and its homologs in other herpesviruses protected viral proteins from host autophagic signaling, thus facilitating herpesvirus replication.Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CASP3: caspase 3; cl-CASP3: cleaved-CASP3; co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; EHV-1: equine herpesvirus 1; gM: glycoprotein M; HCMV: human cytomegalovirus; HSV-1: herpes simplex virus 1; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; OD: optical density; PCR: polymerase chain reaction; PFU: plaque forming units; PRV: pseudorabies virus; Rap: rapamycin; SNAP29; synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TCID: 50% tissue culture infectious doses; UBA: ubiquitin-binding domain; VAMP8: vesicle associated membrane protein 8; µm, micrometer; VZV: varicella-zoster virus; WT: wild type.
African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a devastating disease epidemic in Asia and Europe. Large knowledge gaps regarding the biological characteristics of viral structural proteins have severely hindered the development of vaccines against ASF. The p22 protein, an internal envelope membrane protein of ASFV, is one such protein that is yet to be deciphered despite its significance. Here, our results indicated that p22 is not essential for the morphogenesis and replication of ASFV in porcine alveolar macrophages. The ASFV p22 negatively regulates the IFN-β-triggered activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Mechanistically, the ASFV p22 promotes the association of the Tax1-binding protein 1 (TAX1BP1) with the type I IFN receptor 1 (IFNAR1) via its transmembrane region, thereby facilitating the autophagic degradation of IFNAR1 and impairing the host antiviral responses at the initial step of JAK-STAT signaling pathway. These findings clarify the biological functions of p22 in ASFV replication and uncover a novel autophagy degradation mechanism for IFNAR1, which provide a novel theoretical basis for understanding the biological characteristics of ASFV and may contribute to the development of vaccines and antiviral therapies against ASF.
The transmission cycle of Japanese encephalitis virus (JEV), involving pigs and birds as amplifying hosts and mosquitoes as vectors, was elucidated in the 1950s. However, factors contributing to this cycle remain unclear. Here, sheep were infected with a JEV strain isolated from sheep exhibiting neurological symptoms. The results revealed that sheep are susceptible to JEV infection and develop viremia, with levels and duration comparable to those observed in pigs, a known JEV-amplifying host. Mosquitoes fed viremic sheep blood showed an infection rate of 40.6 to 57.1%. These findings indicate that sheep can serve as amplifying hosts for JEV, potentially contributing to JEV transmission and increasing the public health risk of human infections. We propose an alternative, sheep-associated rural domestic JEV transmission cycle, which may be prevalent in specific regions where sheep are bred but pigs are not. This cycle exists along with the well-known pig-associated rural domestic and bird-associated wild cycles.
Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu stricto (s.s.), poses a substantial risk to both humans and domestic animals globally. Here, we compared the whole genomes of 111 E. granulosus s.s. samples from China. Genomic variation data revealed frequent cross-fertilization in the hermaphroditic E. granulosus. The G1 and G3 genotypes represent distinct mitochondrial lineages, while showing no differentiation in the nuclear genome, suggesting mito-nuclear discordance caused by historical geographic separation and subsequent fusion. Population structure, demographic history, and gene flow among populations reflected the transmission route of E. granulosus s.s. from the Middle East to Qinghai-Xizang Plateau through the migration of nomadic people, followed by introgression during secondary contact. Genomic variations highlighted selection signatures within the genome prone to balancing selection, particularly impacting genes encoding membrane-related proteins, representing a potential evolutionary strategy for adaptation to parasitic life. Balancing selection pressure on the gene-coding sodium/bile acid cotransporter led to its high level of genetic stability, which may play a crucial role in the survival and development of E. granulosus during the parasitic stage, making it a potential drug target for the treatment of CE. Meanwhile, other genomic regions under strong balancing selection may provide potential targets for protective immunity. These findings offer valuable insights into the complex dynamics and adaptive evolution of E. granulosus s.s. in China.IMPORTANCEEchinococcus granulosus sensu stricto (s.s.) is the primary cause of cystic echinococcosis (CE), a parasitic disease affecting humans and livestock with significant health and economic impacts. Previous studies on this parasite relied on mitochondrial DNA to classify its genotypes and understand its genetic diversity. However, these studies cannot capture the full complexity of its evolutionary dynamics and adaptation strategies. Our research employs comprehensive genome-wide sequencing, offering a more nuanced view of its genetic landscape. We discovered that cross-fertilization appears to be a prevalent reproductive strategy in the hermaphroditic E. granulosus, underpinning the observed deep mitochondrial divergence between genotypes G1 and G3, as well as gene flow among populations. The transmission history of E. granulosus s.s. in China and its widespread genetic mixing were likely facilitated by the migrations of nomadic peoples. Furthermore, we identified genes under balancing selection, including the gene involved in the uptake of host bile acids, which play a crucial role in the parasite's survival and development, potentially offering new targets for intervention. Our research advances the understanding of the genetic diversity and evolutionary strategies of E. granulosus, laying the foundation for improved control measures of CE.
The Porcine Epidemic Diarrhea Virus (PEDV) is one of the major challenges facing the global pig farming industry, and vaccines and treatments have proven difficult in controlling its spread. Faecalibacterium prausnitzii (F.prausnitzii), a key commensal bacterium in the gut, has been recognized as a promising candidate for next-generation probiotics due to its potential wide-ranging health benefits. A decrease in F.prausnitzii abundance has been associated with certain viral infections, suggesting its potential application in preventing intestinal viral infections. In this study, we utilized a piglet model to examine the potential role of F.prausnitzii in PEDV infections. A piglet model of PEDV infection was established and supplemented with F.prausnitzii, revealing that F.prausnitzii mitigated PEDV infection. Further studies found that outer membrane vesicles (OMVs) are the main functional components of F.prausnitzii, and proteomics, untargeted metabolomics, and small RNA-seq were used to analyze the composition of OMVs. Exhaustion of the gut microbiota demonstrated that the function of Fp. OMVs relies on the presence of the gut microbiota. Additionally, metagenomic analysis indicated that Fp. OMVs altered the gut microbiota composition, enhancing the abundance of Faecalibacterium prausnitzii, Prevotellamassilia timonensis, and Limosilactobacillus reuteri. Untargeted metabolomics analysis showed that Fp. OMVs increased phosphatidylcholine (PC) levels, with PC identified as a key metabolite in alleviating PEDV infection. Single-cell sequencing revealed that PC altered the relative abundance of intestinal cells, increased the number of intestinal epithelial cells, and reduced necroptosis in target cells. PC treatment in infected IPEC-J2 and Vero cells alleviated necroptosis and reduced the activation of the RIPK1-RIPK3-MLKL signaling axis, thereby improving PEDV infection. F.prausnitzii and its OMVs play a critical role in mitigating PEDV infections. These findings provide a promising strategy to ameliorate PEDV infection in piglets.
Peste des petits ruminants virus (PPRV) is an important pathogen that has long been a significant threat to small ruminant productivity worldwide. Iron metabolism is vital to the host and the pathogen. However, the mechanism underlying host-PPRV interactions from the perspective of iron metabolism and iron-mediated membrane lipid peroxidation has not been reported thus far. In this study, we identified a novel host long-noncoding RNA (lncRNA), APR, that impairs PPRV infectivity by sponging miR-3955-5p, a negative microRNA (miRNA) that directly targets the gene encoding the ferritin-heavy chain 1 (FTH1) protein. Importantly, we demonstrated that PPRV infection causes aberrant cellular iron accumulation by increasing transferrin receptor (TFRC) expression and that iron accumulation induces reticulophagy and ferroptosis, which benefits PPRV replication. Moreover, PPRV infection enhanced the localization of cellular iron on the endoplasmic reticulum (ER) and caused ER membrane damage by promoting excess lipid peroxidation to induce reticulophagy. Interestingly, APR decreased PPRV infection-induced accumulation of intracellular Fe2+ via miR-3955-5p/FTH1 axis and ultimately inhibited reticulophagy and ferroptosis. Additionally, our results indicate that interferon regulatory factor 1 promotes APR transcription by positively regulating APR promoter activity after PPRV infection. Taken together, our findings revealed a new pattern of PPRV-host interactions, involving noncoding RNA regulation, iron metabolism, and iron-related membrane lipid peroxidation, which is critical for understanding the host defense against PPRV infection and the pathogenesis of PPRV.IMPORTANCEMany viruses have been demonstrated to engage in iron metabolism to facilitate their replication and pathogenesis. However, the mechanism by which PPRV interacts with host cells from the perspective of iron metabolism, or iron-mediated membrane lipid peroxidation, has not yet been reported. Our data provide the first direct evidence that PPRV infection induces aberrant iron accumulation to promote viral replication and reveal a novel host lncRNA, APR, as a regulator of iron accumulation by promoting FTH1 protein expression. In this study, PPRV infection increased cellular iron accumulation by increasing TFRC expression, and more importantly, iron overload increased viral infectivity as well as promoted ER membrane lipid peroxidation by enhancing the localization of cellular iron on the ER and ultimately induced ferroptosis and reticulophagy. Furthermore, a host factor, the lncRNA APR, was found to decrease cellular iron accumulation by sponging miR-3955-5p, which directly targets the gene encoding the FTH1 protein, thereby attenuating PPRV infection-induced ferroptosis and reticulophagy and inhibiting PPRV infection. Taken together, the results of the present study provide new insight into our understanding of host-PPRV interaction and pathogenesis from the perspective of iron metabolism and reveal potential targets for therapeutics against PPRV infection.
N6-methyladnosine (m6A) modification is present in both positive- and negative-strand RNA of influenza A virus (IAV) and affects the replication and pathogenicity of IAV. However, little is known about the regulatory mechanism of m6A in IAV RNA. In the present study, we identified the m6A methylation of the viral RNA of different IAV subtypes and confirmed that m6A modification promotes the polymerase activity and replication of IAV. By mutating m6A motifs on the multiple viral RNAs (vRNAs) of IAV, we revealed that m6A deficiency in vRNA suppresses the expression of viral genes and the replication of the virus in vitro. In addition, m6A deficiency in vRNA reduced the pathogenicity of IAV in a mouse model. Mechanistically, m6A deficiency in vRNA suppresses the assembly of the viral ribonucleoprotein (vRNP) complex by impairing the interaction between vRNA and vRNP proteins in an m6A methyltransferase-dependent manner, but not the m6A reader proteins. Together, our findings reveal an important role for m6A on viral RNAs in facilitating the activity of the polymerase complex and the replication and pathogenicity of IAV, which provides insights for the development of novel anti-influenza strategies.
Betacoronavirus is a causative agent of respiratory and enteric diseases in humans and animals. Several ruminants are recognized to be intermediate hosts in the transmission of emerging coronaviruses from reservoir hosts to humans. Here, we first report a novel Betacoronavirus isolated from goats suffering from diarrhea in China, putatively named caprine coronavirus (cpCoV). Full-genome characterization and nuclear acid comparisons demonstrated that this virus is an evolutionarily distinct Betacoronavirus belonging to the subgenus Embecovirus and is a Betacoronavirus 1 species. Notably, on phylogenetic trees based on complete genomes and RdRp, S, and N genes, the cpCoVs were grouped into a clade distinct from other Betacoronavirus strains and were closely related to the HKU23- and HKU23-associated coronaviruses. CpCoV possessed a unique genome organization with a truncated NS4a protein and an elongated NS4b protein that showed no significant matches in the GenBank database. The homology of the S and NS4a-4b genes between cpCoV and Embecovirus was less than 95%. Analysis revealed possible recombination events occurred during the evolution of cpCoV and HKU23, and there are striking similarities between the two viruses in evolutionary terms. In addition, cpCoV showed a narrow cell tropism, replicating in human- and bovine-origin cells in vitro, and caused diarrhea and enteric pathologic changes in goats and calves in vivo. We have provided epidemiological, virological, evolutionary, and experimental evidence that cpCoV is a novel etiological agent for enteric disease in goats. Evidently, a spilling-over event might have occurred between ruminants, including goats, camels, cattle, and wild animals. This study highlights the importance of identifying coronavirus diversity and inter-species transmission in ruminants worldwide, broadens our understanding of the ecology of coronaviruses, and aids in the prevention of animal-to-human transmission and outbreaks.
The endoribonuclease (EndoU) nsp15 of coronaviruses plays a crucial role in evading host innate immune responses by reducing the abundance of viral double-stranded RNA (dsRNA). However, our understanding of its interactions with host cellular targets remains limited. In this study, we demonstrate that overexpression of nsp15 from four coronavirus genera inhibits cellular protein synthesis and causes nuclear retention of PABPC1. Mutation analysis confirms the essential role of EndoU activity in these processes. Fluorescence in situ hybridization (FISH) analysis shows that cellular mRNA co-localizes with nsp15 in certain cells. Real time RT-PCR indicates that the mRNA levels of several antiviral genes decrease in cells expressing nsp15, and this reduction depends on the EndoU activity of nsp15. Using infectious bronchitis virus (IBV) as a model, we investigate the inhibitory effect of nsp15 on protein translation during infection. We find that infection with IBV with functional nsp15 suppresses protein synthesis in a PKR-eIF2α independent manner, with PABPC1 mainly located in the cytoplasm. However, infection with EndoU activity-deficiency mutant virus rIBV-nsp15-H238A results in the accumulation of viral dsRNA, triggering a PKR-eIF2α-dependent shutdown of protein synthesis and leading to the nuclear relocation of PABPC1. In the absence of the PKR-eIF2α pathway, IBV is still able to suppress host protein synthesis, while the inhibitory effect of rIBV-nsp15-H238A on protein synthesis was significantly reduced. Although nsp15 locates to replication-transcription complex (RTC) during infection, RNA immunoprecipitation (RIP)-Seq analysis confirms that IBV nsp15 binds to six viral RNAs and 237 cellular RNAs. The proteins encoded by the nsp15-associated cellular RNAs predominantly involved in translation. Additionally, proteomic analysis of the nsp15 interactome identifies 809 cellular proteins, which are significantly enriched in pathways related to ribosome biogenesis, RNA processing, and translation. Therefore, nsp15 helps virus circumvent the detrimental PKR-eIF2α pathway by reducing viral dsRNA accumulation and suppresses host protein synthesis by targeting host RNAs and proteins. This study reveals unique yet conserved mechanisms of protein synthesis shutdown by catalytically active nsp15 EndoU, shedding light on how coronaviruses regulate host protein expression.
Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes severe diarrhea in neonatal piglets worldwide and presents a significant public health threat due to its potential for cross-species transmission. Selective macroautophagy/autophagy, mediated by autophagy receptors such as NBR1 (NBR1 autophagy cargo receptor), plays a key role in restricting viral infection and modulating the host immune response. In this study, we revealed that overexpression of NBR1 inhibits PDCoV replication, while its knockdown increases viral titers. Further analysis demonstrated that NBR1 interacts with the PDCoV envelope (E) protein independently of ubiquitination, directing it to phagophores for autophagic degradation to limit viral proliferation. To counteract this defense, PDCoV 3C-like protease, encoded by NSP5, cleaves porcine NBR1 at glutamine 353 (Q353), impairing its selective autophagy function and antiviral activity. Additionally, we demonstrated that NSP5 proteases from other coronaviruses including PEDV, TGEV, and SARS-CoV-2 also cleave NBR1 at the same site, suggesting that coronaviruses employ a conserved strategy of NSP5-mediated cleavage of NBR1 to evade host antiviral responses and facilitate infection. Overall, our study underscores the importance of NBR1-mediated selective autophagy in the host's defense against PDCoV and reveals a strategy by which PDCoV evades autophagic mechanisms to promote successful infection.Abbreviation: Cas9: CRISPR-associated protein 9; CC1: coiled-coil 1; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; KO: knockout; LIR: MAP1LC3/LC3-interacting region; mAb: monoclonal antibody; NBR1: NBR1 autophagy cargo receptor; NBR1-C: C-terminal fragment of NBR1; NBR1-N: N-terminal fragment of NBR1; OPTN: optineurin; pAb: polyclonal antibody; PB1: Phox/BEM1 domain; PDCoV: porcine deltacoronavirus; PEDV: porcine epidemic diarrhea virus; Q353A: a NBR1 construct with the glutamine (Q) residue at position 353 replaced with glutamic acid (A); SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1: sequestosome 1; TCID50: 50% tissue culture infective dose; TGEV: porcine transmissible gastroenteritis virus; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild type; ZZ: ZZ-type zinc finger domain.
The H9N2 subtype of avian influenza virus (AIV) emerges as a significant member of the influenza A virus family. However, the varying degrees of epidemiological dominance among different lineages or clades of H9N2 AIVs have not been fully clarified. The matrix protein M1, a key structural component of the virion, plays a crucial role in maintaining the viral structure and lifecycle. To elucidate the intrinsic relationship between the genetic patterns of M1 and the adaptive dynamics of H9N2 AIVs, this study focused on the five major evolutionary patterns of M1 and conducted in vitro and in vivo investigations from the perspectives of vRNP release after viral uncoating, polymerase activity, mRNA and vRNA levels, the nuclear export of vRNPs, plasma membrane-binding capacity, proliferation capacity, growth competitiveness, and transmission potential. The results revealed a strong correlation between the epidemiological dominance of H9N2 AIVs and the specific patterns of M1, with M1P5 standing out as particularly significant. This finding highlights the pivotal influence of the M1 gene patterns on the replication and transmission dynamics of H9N2 AIVs, thereby offering valuable insights into the mechanisms driving differences in adaptive evolution and shifts in epidemiological dominance within the H9N2 AIV population.
Alveolar echinococcosis (AE) is a severe and life-threatening parasitic disease caused by Echinococcus multilocularis. Liver fibrosis is a significant pathological feature of advanced AE, characterized by the excessive production and accumulation of extracellular matrix (ECM). However, the precise underlying mechanism remains largely unknown. In this study, we show that the long noncoding RNA gm40262, predominantly expressed in hepatic stellate cells (HSCs), is upregulated in AE. Interestingly, its knockdown leads to liver fibrosis resolution, accompanied by a substantial suppression of parasite growth. Gm40262 functions by targeting miR-193b-5p to activate HSCs and stimulate their proliferation in a TGF-β-dependent manner, thereby promoting ECM production by upregulating Col1α1. Moreover, gm40262 is also involved in inflammation through the gm40262-miR-193b-5p-TLR4 axis. Our findings suggest that gm40262 plays a pivotal role in parasite-induced liver fibrosis through multiple mechanisms, highlighting its potential as a therapeutic target for hepatic fibrosis. IMPORTANCE:Echinococcus multilocularis is a tiny parasite with significant medical implications. The chronic parasitism of E. multilocularis in the liver generally leads to liver fibrosis, but the underlying mechanisms are poorly understood. We herein show that gm40262, a long noncoding RNA predominantly expressed in hepatic stellate cells (HSCs), is involved in hepatic fibrogenesis during infection by activating HSCs and promoting extracellular matrix production. The gm40262-orchestrating fibrogenesis occurs through the gm40262-miR-193b-5p-TLR4 and gm40262-miR-193b-5p-Col1α1 axes. The knockdown of gm40262 remarkably alleviates liver fibrosis, with decreased parasite growth. Our findings reveal a key role of gm40262 in liver fibrosis during E. multilocularis infection, rendering it a therapeutic target for hepatic fibrosis.
Despite decades of research on effective methods to resist Salmonella enterica serovar Typhimurium (S. Typhimurium) pathogenicity, the mechanisms of S. Typhimurium-host interactions have not been fully determined. S. Typhimurium is characterized as an important zoonosis in public health worldwide because of its endemicity, high morbidity, and difficulty in applying control and prevention measures. Herein, we introduce a novel bacterial factor, secretion system effector J (SseJ), and its interactive host protein, PHB2 (prohibitin 2). We explored whether SseJ affected S. Typhimurium replication and survival in the host. S. Typhimurium infection caused severe mitochondrial damage and mitophagy, which facilitated S. Typhimurium proliferation in cells. S. Typhimurium SseJ activated the PINK1 (PTEN induced kinase 1)-PRKN (parkin RBR E3 ubiquitin protein ligase)-autophagosome-dependent mitophagy pathway, aided by the mitophagy receptor PHB2, for bacterial survival and persistent infection. Moreover, suppression of mitophagy alleviated the pathogenicity of S. Typhimurium. In conclusion, S. Typhimurium infection could be antagonized by targeting the SseJ-PHB2-mediated host mitochondrial autophagy pathway.Abbreviation: ACTB: actin beta; BafA1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; co-IP: co-immunoprecipitation; CFU: colony-forming units; COX4/COXIV: cytochrome c oxidase subunit 4; CQ: chloroquine; hpi: h post-bacterial infection; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Mdivi-1:mitophagy inhibitor mitochondrial division inhibitor 1; MFN2: mitofusin 2; MG132: z-leu-leu-leucinal; MOI: multiplicity of infection; mtDNA: mitochondrial DNA; PBS: phosphate-buffered saline; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; qPCR: quantitative real-time reverse transcription PCR; Roc-A: Rocaglamide A; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; SCVs: Salmonella-containing vacuoles; siRNA: small interfering RNA; SPI-2: Salmonella pathogenicity island 2; SseJ: secretion system effector J; S. Typhimurium: Salmonella enterica serovar Typhimurium; S.T-ΔSseJ: SseJ gene-deleted Salmonella Typhimurium strains; S.T-CΔSseJ: SseJ-complemented Salmonella Typhimurium strains; WT: wild-type.
Bacteriophages are promising alternatives for combating multidrug-resistant bacterial infections. Two lytic bacteriophages, named P1 and P3, targeting pathogenic Escherichia coli (ExPEC; strain TZ1_3) were isolated and evaluated for their potential ability to control pathogenic numbers either in ExPEC-contaminated food or ExPEC-infected mice. Results showed that phages significantly reduced ExPEC numbers within 6 and 12 h in contaminated water, milk, beef, and chicken when applied at 106 plaque-forming units (PFU). Notably, phage therapy administered via intraperitoneal injection (1012 PFU) effectively reduced ExPEC numbers in the heart, liver, spleen and kidney, restored α-diversity of gut microbes, and increased levels of acetic (13.98 %-37.58 %) and valeric acid (10.27 %-31.51 %) in ExPEC-infected mice. Additionally, phage injections caused no detrimental effects on body weight (which increased by 6.49 %-8.11 %), and on gut microbes in healthy mice. Overall, this study highlights the potential of phages in controlling foodborne microorganisms.
Porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis coronavirus (TGEV), the two causative agents of porcine diarrhea, have been reported to be at risk of cross-species transmission, including to humans. However, the potential host range in which these two CoVs interact remains unclear. We screened 16 animal counterparts for porcine aminopeptidase N (APN), the receptor of PDCoV and TGEV, and found that APNs from eight of 17 animals could bind to the receptor-binding domains (RBDs) of PDCoV and TGEV. Furthermore, the animal APNs that could bind to the RBDs could mediate cellular infection by both viruses. Dog APN (dAPN) has been identified as the animal receptor with the highest capability to mediate the virus infection. We further resolved the complex structures of dAPN bound to the PDCoV RBD/TGEV RBD, respectively, establishing its divergent receptor-binding modes. We identified R325 of dAPN as an important residue in the PDCoV RBD-dAPN interaction, and found the central role of Q746 and T749 in dAPN in the interaction with the TGEV RBD. These findings provide the molecular basis of the potential cross-species transmission of these two porcine CoVs and shed light on future surveillance of these CoVs.
