ABSTRACT
Mitochondria are energy producers in cells, which can affect viral replication by regulating the host innate immune signaling pathways, and the changes in their biological functions are inextricably linked the viral life cycle. In this study, we screened a library of 382 mitochondria-targeted compounds and identified the antiviral inhibitors of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo synthesis pathway of pyrimidine ribonucleotides, against classical swine fever virus (CSFV). Our data showed that the inhibitors interfered with viral RNA synthesis in a dose-dependent manner, with half-maximal effective concentrations (EC50) ranging from 0.975 to 26.635 nM. Remarkably, DHODH inhibitors obstructed CSFV replication by enhancing the innate immune response including the TBK1-IRF3-STAT1 and NF-κB signaling pathways. Furthermore, the data from a series of compound addition and supplementation trials indicated that DHODH inhibitors also inhibited CSFV replication by blocking the de novo pyrimidine synthesis. Remarkably, DHODH knockdown demonstrated that it was essential for CSFV replication. Mechanistically, confocal microscopy and immunoprecipitation assays showed that the non-structural protein 4A (NS4A) recruited and interacted with DHODH in the perinuclear. Notably, NS4A enhanced the DHODH activity and promoted the generation of UMP for efficient viral replication. Structurally, the amino acids 65-229 of DHODH and the amino acids 25-40 of NS4A were pivotal for this interaction. Taken together, our findings highlight the critical role of DHODH in the CSFV life cycle and offer a potential antiviral target for the development of novel therapeutics against CSF. IMPORTANCE: Classical swine fever remains one of the most economically important viral diseases of domestic pigs and wild boar worldwide. dihydroorotate dehydrogenase (DHODH) inhibitors have been shown to suppress the replication of several viruses in vitro and in vivo, but the effects on Pestivirus remain unknown. In this study, three specific DHODH inhibitors, including DHODH-IN-16, BAY-2402234, and Brequinar were found to strongly suppress classical swine fever virus (CSFV) replication. These inhibitors target the host DHODH, depleting the pyrimidine nucleotide pool to exert their antiviral effects. Intriguingly, we observed that the non-structural protein 4A of CSFV induced DHODH to accumulate around the nucleus in conjunction with mitochondria. Moreover, NS4A exhibited a strong interaction with DHODH, enhancing its activity to promote efficient CSFV replication. In conclusion, our findings enhance the understanding of the pyrimidine synthesis in CSFV infection and expand the novel functions of CSFV NS4A in viral replication, providing a reference for further exploration of antiviral targets against CSFV.
Subject(s)
Antiviral Agents , Classical Swine Fever Virus , Dihydroorotate Dehydrogenase , Viral Nonstructural Proteins , Virus Replication , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Cell Line , Classical Swine Fever/drug therapy , Classical Swine Fever/immunology , Classical Swine Fever/metabolism , Classical Swine Fever/virology , Classical Swine Fever Virus/drug effects , Classical Swine Fever Virus/growth & development , Classical Swine Fever Virus/immunology , Classical Swine Fever Virus/metabolism , Dihydroorotate Dehydrogenase/metabolism , Dose-Response Relationship, Drug , Immunity, Innate/drug effects , Immunity, Innate/immunology , Immunoprecipitation , Microscopy, Confocal , Mitochondria/enzymology , Mitochondria/metabolism , RNA, Viral/biosynthesis , Signal Transduction/drug effects , Swine/virology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effectsABSTRACT
Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.
Subject(s)
Myxovirus Resistance Proteins , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Viral Nonstructural Proteins , Virus Replication , Animals , Cell Line , Interferons/immunology , Interferons/metabolism , Mutation , Myxovirus Resistance Proteins/chemistry , Myxovirus Resistance Proteins/genetics , Myxovirus Resistance Proteins/metabolism , Porcine Reproductive and Respiratory Syndrome/enzymology , Porcine Reproductive and Respiratory Syndrome/metabolism , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/growth & development , Porcine respiratory and reproductive syndrome virus/metabolism , Protein Binding , Swine/virology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolismABSTRACT
Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.
Subject(s)
Chickens , Ducks , Horses , Influenza A virus , Influenza in Birds , Neuraminic Acids , Animals , Humans , Chickens/genetics , Chickens/metabolism , Chickens/virology , Ducks/genetics , Ducks/metabolism , Ducks/virology , Epitopes/chemistry , Epitopes/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Horses/genetics , Horses/metabolism , Horses/virology , Influenza A virus/chemistry , Influenza A virus/classification , Influenza A virus/metabolism , Influenza in Birds/genetics , Influenza in Birds/transmission , Influenza in Birds/virology , Mutation , N-Acetylneuraminic Acid/chemistry , N-Acetylneuraminic Acid/metabolism , Neuraminic Acids/chemistry , Neuraminic Acids/metabolism , Receptors, Virus/chemistry , Receptors, Virus/genetics , Receptors, Virus/metabolism , Swine/virology , Viral Zoonoses/metabolism , Viral Zoonoses/transmission , Viral Zoonoses/virologyABSTRACT
Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease, which is responsible for enormous economic losses to the global pig industry. Although vaccination has been used to prevent PRV infection, the effectiveness of vaccines has been greatly diminished with the emergence of PRV variants. Therefore, there is an urgent need to develop anti-PRV drugs. Polyethylenimine (PEI) is a cationic polymer and has a wide range of antibacterial and antiviral activities. This study found that a low dose of 1 µg/mL of the 25-kDa linear PEI had significantly specific anti-PRV activity, which became more intense with increasing concentrations. Mechanistic studies revealed that the viral adsorption stage was the major target of PEI without affecting viral entry, replication stages, and direct inactivation effects. Subsequently, we found that cationic polymers PEI and Polybrene interfered with the interaction between viral proteins and cell surface receptors through electrostatic interaction to exert the antiviral function. In conclusion, cationic polymers such as PEI can be a category of options for defense against PRV. Understanding the anti-PRV mechanism also deepens host-virus interactions and reveals new drug targets for anti-PRV.IMPORTANCEPolyethylenimine (PEI) is a cationic polymer that plays an essential role in the host immune response against microbial infections. However, the specific mechanisms of PEI in interfering with pseudorabies virus (PRV) infection remain unclear. Here, we found that 25-kDa linear PEI exerted mechanisms of antiviral activity and the target of its antiviral activity was mainly in the viral adsorption stage. Correspondingly, the study demonstrated that PEI interfered with the virus adsorption stage by electrostatic adsorption. In addition, we found that cationic polymers are a promising novel agent for controlling PRV, and its antiviral mechanism may provide a strategy for the development of antiviral drugs.
Subject(s)
Antiviral Agents , Herpesvirus 1, Suid , Polyethyleneimine , Static Electricity , Animals , Adsorption/drug effects , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Herpesvirus 1, Suid/drug effects , Herpesvirus 1, Suid/metabolism , Polyethyleneimine/chemistry , Polyethyleneimine/pharmacology , Pseudorabies/drug therapy , Pseudorabies/virology , Swine/virology , Swine Diseases/virologyABSTRACT
African swine fever (ASF) is a highly contagious viral disease that affects domestic and wild pigs. The causative agent of ASF is African swine fever virus (ASFV), a large double-stranded DNA virus with a complex virion structure. Among the various proteins encoded by ASFV, A137R is a crucial structural protein associated with its virulence. However, the structure and molecular mechanisms underlying the functions of A137R remain largely unknown. In this study, we present the structure of A137R determined by cryogenic electron microscopy single-particle reconstruction, which reveals that A137R self-oligomerizes to form a dodecahedron-shaped cage composed of 60 polymers. The dodecahedron is literally equivalent to a T = 1 icosahedron where the icosahedral vertexes are located in the center of each dodecahedral facet. Within each facet, five A137R protomers are arranged in a head-to-tail orientation with a long N-terminal helix forming the edge through which adjacent facets stitch together to form the dodecahedral cage. Combining structural analysis and biochemical evidence, we demonstrate that the N-terminal domain of A137R is crucial and sufficient for mediating the assembly of the dodecahedron. These findings imply the role of A137R cage as a core component in the icosahedral ASFV virion and suggest a promising molecular scaffold for nanotechnology applications. IMPORTANCE: African swine fever (ASF) is a lethal viral disease of pigs caused by African swine fever virus (ASFV). No commercial vaccines and antiviral treatments are available for the prevention and control of the disease. A137R is a structural protein of ASFV that is associated with its virulence. The discovery of the dodecahedron-shaped cage structure of A137R in this study is of great importance in understanding ASFV pathogenicity. This finding sheds light on the molecular mechanisms underlying the functions of A137R. Furthermore, the dodecahedral cage formed by A137R shows promise as a molecular scaffold for nanoparticle vectors. Overall, this study provides valuable insights into the structure and function of A137R, contributing to our understanding of ASFV and potentially opening up new avenues for the development of vaccines or treatments for ASF.
Subject(s)
African Swine Fever Virus , Swine , Viral Structural Proteins , Animals , African Swine Fever/virology , African Swine Fever Virus/chemistry , African Swine Fever Virus/growth & development , African Swine Fever Virus/pathogenicity , African Swine Fever Virus/ultrastructure , Cryoelectron Microscopy , Structure-Activity Relationship , Swine/virology , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolism , Viral Structural Proteins/ultrastructure , Virion/chemistry , Virion/metabolism , Virion/ultrastructure , VirulenceABSTRACT
United States and other countries may soon approve virus-proof pigs.
Subject(s)
Animals, Genetically Modified , Disease Resistance , Gene Editing , Meat , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Animals , Swine/genetics , Swine/virology , Animals, Genetically Modified/genetics , Animals, Genetically Modified/virology , United States , Porcine Reproductive and Respiratory Syndrome/genetics , Disease Resistance/geneticsABSTRACT
Viruses exploit the host cell's energy metabolism system to support their replication. Mitochondria, known as the powerhouse of the cell, play a critical role in regulating cell survival and virus replication. Our prior research indicated that the classical swine fever virus (CSFV) alters mitochondrial dynamics and triggers glycolytic metabolic reprogramming. However, the role and mechanism of PKM2, a key regulatory enzyme of glycolytic metabolism, in CSFV replication remain unclear. In this study, we discovered that CSFV enhances PKM2 expression and utilizes PKM2 to inhibit pyruvate production. Using an affinity purification coupled mass spectrometry system, we successfully identified PKM as a novel interaction partner of the CSFV non-structural protein NS4A. Furthermore, we validated the interaction between PKM2 and both CSFV NS4A and NS5A through co-immunoprecipitation and confocal analysis. PKM2 was found to promote the expression of both NS4A and NS5A. Moreover, we observed that PKM2 induces mitophagy by activating the AMPK-mTOR signaling pathway, thereby facilitating CSFV proliferation. In summary, our data reveal a novel mechanism whereby PKM2, a metabolic enzyme, promotes CSFV proliferation by inducing mitophagy. These findings offer a new avenue for developing antiviral strategies. IMPORTANCE: Viruses rely on the host cell's material-energy metabolic system for replication, inducing host metabolic disorders and subsequent immunosuppression-a major contributor to persistent viral infections. Classical swine fever virus (CSFV) is no exception. Classical swine fever is a severe acute infectious disease caused by CSFV, resulting in significant economic losses to the global pig industry. While the role of the metabolic enzyme PKM2 (pyruvate dehydrogenase) in the glycolytic pathway of tumor cells has been extensively studied, its involvement in viral infection remains relatively unknown. Our data unveil a new mechanism by which the metabolic enzyme PKM2 mediates CSFV infection, offering novel avenues for the development of antiviral strategies.
Subject(s)
AMP-Activated Protein Kinases , Classical Swine Fever Virus , Mitophagy , Pyruvate Kinase , TOR Serine-Threonine Kinases , Viral Nonstructural Proteins , Virus Replication , Animals , AMP-Activated Protein Kinases/metabolism , Antiviral Agents , Classical Swine Fever/metabolism , Classical Swine Fever/virology , Classical Swine Fever Virus/growth & development , Classical Swine Fever Virus/physiology , Drug Design , Glycolysis , Pyruvate Kinase/chemistry , Pyruvate Kinase/metabolism , Pyruvates/metabolism , Signal Transduction , Swine/metabolism , Swine/virology , TOR Serine-Threonine Kinases/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolismABSTRACT
Porcine deltacoronavirus (PDCoV) has caused enormous economic losses to the global pig industry. However, the immune escape mechanism of PDCoV remains to be fully clarified. Transcriptomic analysis revealed a high abundance of interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) transcripts after PDCoV infection, which initially implied a correlation between IFIT3 and PDCoV. Further studies showed that PDCoV nsp5 could antagonize the host type I interferon signaling pathway by cleaving IFIT3. We demonstrated that PDCoV nsp5 cleaved porcine IFIT3 (pIFIT3) at Gln-406. Similar cleavage of endogenous IFIT3 has also been observed in PDCoV-infected cells. The pIFIT3-Q406A mutant was resistant to nsp5-mediated cleavage and exhibited a greater ability to inhibit PDCoV infection than wild-type pIFIT3. Furthermore, we found that cleavage of IFIT3 is a common characteristic of nsp5 proteins of human coronaviruses, albeit not alphacoronavirus. This finding suggests that the cleavage of IFIT3 is an important mechanism by which PDCoV nsp5 antagonizes IFN signaling. Our study provides new insights into the mechanisms by which PDCoV antagonizes the host innate immune response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a potential emerging zoonotic pathogen, and studies on the prevalence and pathogenesis of PDCoV are ongoing. The main protease (nsp5) of PDCoV provides an excellent target for antivirals due to its essential and conserved function in the viral replication cycle. Previous studies have revealed that nsp5 of PDCoV antagonizes type I interferon (IFN) production by targeting the interferon-stimulated genes. Here, we provide the first demonstration that nsp5 of PDCoV antagonizes IFN signaling by cleaving IFIT3, which affects the IFN response after PDCoV infection. Our findings reveal that PDCoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by deltacoronaviruses.
Subject(s)
Coronavirus 3C Proteases , Coronavirus Infections , Deltacoronavirus , Interferon Type I , Intracellular Signaling Peptides and Proteins , Swine Diseases , Swine , Animals , Humans , Coronavirus 3C Proteases/metabolism , Coronavirus Infections/immunology , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Deltacoronavirus/enzymology , Deltacoronavirus/metabolism , Deltacoronavirus/pathogenicity , Immunity, Innate , Interferon Type I/antagonists & inhibitors , Interferon Type I/biosynthesis , Interferon Type I/immunology , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Proteolysis , Signal Transduction/immunology , Swine/immunology , Swine/virology , Swine Diseases/immunology , Swine Diseases/metabolism , Swine Diseases/virology , Transcription Factors/metabolism , Viral Zoonoses/immunology , Viral Zoonoses/virology , Virus ReplicationABSTRACT
African swine fever virus (ASFV) causes a highly contagious and deadly disease in domestic pigs and European wild boars, posing a severe threat to the global pig industry. ASFV CP204L, a highly immunogenic protein, is produced during the early stages of ASFV infection. However, the impact of CP204L protein-interacting partners on the outcome of ASFV infection is poorly understood. To accomplish this, coimmunoprecipitation and mass spectrometry analysis were conducted in ASFV-infected porcine alveolar macrophages (PAMs). We have demonstrated that sorting nexin 32 (SNX32) is a CP204L-binding protein and that CP204L interacted and colocalized with SNX32 in ASFV-infected PAMs. ASFV growth and replication were promoted by silencing SNX32 and suppressed by overexpressing SNX32. SNX32 degraded CP204L by recruiting the autophagy-related protein Ras-related protein Rab-1b (RAB1B). RAB1B overexpression inhibited ASFV replication, while knockdown of RAB1B had the opposite effect. Additionally, RAB1B, SNX32, and CP204L formed a complex upon ASFV infection. Taken together, this study demonstrates that SNX32 antagonizes ASFV growth and replication by recruiting the autophagy-related protein RAB1B. This finding extends our understanding of the interaction between ASFV CP204L and its host and provides new insights into exploring the relationship between ASFV infection and autophagy.IMPORTANCEAfrican swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality near 100% in domestic pigs. ASF virus (ASFV), which is the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size, encoding more than 150 proteins. Currently, there are no available vaccines against ASFV. ASFV CP204L represents the most abundantly expressed viral protein early in infection and plays an important role in regulating ASFV replication. However, the mechanism by which the interaction between ASFV CP204L and host proteins affects ASFV replication remains unclear. In this study, we demonstrated that the cellular protein SNX32 interacted with CP204L and degraded CP204L by upregulating the autophagy-related protein RAB1B. In summary, this study will help us understand the interaction mechanism between CP204L and its host upon infection and provide new insights for the development of vaccines and antiviral drugs.
Subject(s)
African Swine Fever Virus , African Swine Fever , Antiviral Restriction Factors , Autophagy , Sorting Nexins , rab1 GTP-Binding Proteins , Animals , Autophagy-Related Proteins/metabolism , Sus scrofa/virology , Swine/virology , Sorting Nexins/metabolism , Antiviral Restriction Factors/metabolism , rab1 GTP-Binding Proteins/metabolism , Macrophages/virology , Virus ReplicationABSTRACT
A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.
Subject(s)
Macrophages, Alveolar , Pluripotent Stem Cells , Swine , Animals , Endocytosis , Hematopoiesis/drug effects , Lipopolysaccharides/pharmacology , Macrophages, Alveolar/cytology , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/virology , Mesoderm/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Porcine respiratory and reproductive syndrome virus/physiology , Signal Transduction/drug effects , Swine/virology , Transcription Factors/metabolism , Transforming Growth Factor beta/metabolism , Time FactorsABSTRACT
IMPORTANCE: Spike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Cat Diseases , Chiroptera , Dog Diseases , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , Cats , Dogs , Mice , Angiotensin-Converting Enzyme 2/metabolism , Animals, Wild/virology , Cat Diseases/virology , Chiroptera/virology , COVID-19/metabolism , Dog Diseases/virology , Horses/virology , Mutation , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Swine/virology , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
IMPORTANCE: Coronaviruses are important pathogens of humans and animals, and vaccine developments against them are imperative. Due to the ability to induce broad and prolonged protective immunity and the convenient administration routes, live attenuated vaccines (LAVs) are promising arms for controlling the deadly coronavirus infections. However, potential recombination events between vaccine and field strains raise a safety concern for LAVs. The porcine epidemic diarrhea virus (PEDV) remodeled TRS (RMT) mutant generated in this study replicated efficiently in both cell culture and in pigs and retained protective immunogenicity against PEDV challenge in pigs. Furthermore, the RMT PEDV was resistant to recombination and genetically stable. Therefore, RMT PEDV can be further optimized as a backbone for the development of safe LAVs.
Subject(s)
Coronavirus Infections , Porcine epidemic diarrhea virus , Recombination, Genetic , Swine Diseases , Swine , Vaccines, Attenuated , Viral Vaccines , Animals , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/growth & development , Porcine epidemic diarrhea virus/immunology , Swine/immunology , Swine/virology , Swine Diseases/immunology , Swine Diseases/prevention & control , Swine Diseases/virology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology , Virus Replication , Cells, Cultured , MutationABSTRACT
IMPORTANCE: Betacoronaviruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. However, whether all betacoronaviruses members use the same pathway to exit cells remains unknown. Here, we demonstrated that porcine hemagglutinating encephalomyelitis virus (PHEV) egress occurs by Arl8b-dependent lysosomal exocytosis, a cellular egress mechanism shared by SARS-CoV-2 and MHV. Notably, PHEV acidifies lysosomes and activates lysosomal degradative enzymes, while SARS-CoV-2 and MHV deacidify lysosomes and limit the activation of lysosomal degradative enzymes. In addition, PHEV release depends on V-ATPase-mediated lysosomal pH. Furthermore, this is the first study to evaluate ßCoV using lysosome for spreading through the body, and we have found that lysosome played a critical role in PHEV neural transmission and brain damage caused by virus infection in the central nervous system. Taken together, different betacoronaviruses could disrupt lysosomal function differently to exit cells.
Subject(s)
Betacoronavirus 1 , Coronavirus Infections , Exocytosis , Lysosomes , Neurons , Animals , Mice , Betacoronavirus 1/metabolism , Lysosomes/enzymology , Lysosomes/metabolism , Lysosomes/virology , Murine hepatitis virus/metabolism , Neurons/enzymology , Neurons/metabolism , Neurons/pathology , Neurons/virology , SARS-CoV-2/metabolism , Swine/virology , Hydrogen-Ion Concentration , Vacuolar Proton-Translocating ATPases/metabolism , Coronavirus Infections/pathology , Coronavirus Infections/transmission , Coronavirus Infections/virologyABSTRACT
IMPORTANCE: Of the flaviviruses, only CSFV and bovine viral diarrhea virus express Npro as the non-structural protein which is not essential for viral replication but functions to dampen host innate immunity. We have deciphered a novel mechanism with which CSFV uses to evade the host antiviral immunity by the N-terminal domain of its Npro to facilitate proteasomal degradation of Sp1 with subsequent reduction of HDAC1 and ISG15 expression. This is distinct from earlier findings involving Npro-mediated IRF3 degradation via the C-terminal domain. This study provides insights for further studies on how HDAC1 plays its role in antiviral immunity, and if and how other viral proteins, such as the core protein of CSFV, the nucleocapsid protein of porcine epidemic diarrhea virus, or even other coronaviruses, exert antiviral immune responses via the Sp1-HDAC1 axis. Such research may lead to a deeper understanding of viral immune evasion strategies as part of their pathogenetic mechanisms.
Subject(s)
Classical Swine Fever Virus , Classical Swine Fever , Endopeptidases , Histone Deacetylase 1 , Immunity, Innate , Proteasome Endopeptidase Complex , Sp1 Transcription Factor , Viral Proteins , Animals , Classical Swine Fever/immunology , Classical Swine Fever/metabolism , Classical Swine Fever/virology , Classical Swine Fever Virus/enzymology , Classical Swine Fever Virus/immunology , Classical Swine Fever Virus/metabolism , Classical Swine Fever Virus/pathogenicity , Endopeptidases/chemistry , Endopeptidases/metabolism , Histone Deacetylase 1/biosynthesis , Histone Deacetylase 1/metabolism , Interferon Regulatory Factor-3 , Nucleocapsid Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Sp1 Transcription Factor/metabolism , Swine/virology , Viral Core Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Ubiquitins/metabolism , Cytokines/metabolism , Porcine epidemic diarrhea virus/immunology , Porcine epidemic diarrhea virus/metabolism , Protein DomainsABSTRACT
IMPORTANCE: African swine fever virus (ASFV) causes a lethal disease of pigs with high economic impact in affected countries in Africa, Europe, and Asia. The virus encodes proteins that inhibit host antiviral defenses, including the type I interferon response. Host cells also activate cell death through a process called apoptosis to limit virus replication. We showed that the ASFV A179L protein, a BCL-2 family apoptosis inhibitor, is important in reducing apoptosis in infected cells since deletion of this gene increased cell death and reduced virus replication in cells infected with the A179L gene-deleted virus. Pigs immunized with the BeninΔA179L virus showed no clinical signs and a weak immune response but were not protected from infection with the deadly parental virus. The results show an important role for the A179L protein in virus replication in macrophages and virulence in pigs and suggest manipulation of apoptosis as a possible route to control infection.
Subject(s)
African Swine Fever Virus , African Swine Fever , Apoptosis , Gene Deletion , Macrophages , Proto-Oncogene Proteins c-bcl-2 , Swine , Viral Proteins , Virulence , Animals , African Swine Fever/virology , African Swine Fever Virus/genetics , Macrophages/virology , Proto-Oncogene Proteins c-bcl-2/deficiency , Proto-Oncogene Proteins c-bcl-2/genetics , Swine/virology , Virulence/genetics , Virus Replication , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Viral Proteins/geneticsABSTRACT
IMPORTANCE: As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.
Subject(s)
Coronavirus Infections , Coronavirus Nucleocapsid Proteins , Deltacoronavirus , Interferon Type I , Mitochondrial Proteins , Phosphoprotein Phosphatases , Proteolysis , Swine Diseases , Swine , Virus Replication , Animals , Coronavirus Infections/immunology , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Interferon Type I/immunology , Signal Transduction , Swine/virology , Swine Diseases/virology , Ubiquitin-Protein Ligases/metabolism , Virus Replication/immunology , Coronavirus Nucleocapsid Proteins/metabolism , Deltacoronavirus/immunology , Deltacoronavirus/metabolism , Phosphoprotein Phosphatases/metabolism , Mitochondrial Proteins/metabolism , Down-Regulation , Immune Evasion , RNA-Binding Proteins/metabolismABSTRACT
IMPORTANCE: African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines.
Subject(s)
African Swine Fever Virus , African Swine Fever , Interferon Type I , Membrane Proteins , Nucleotides, Cyclic , Swine , Viral Proteins , Animals , African Swine Fever/immunology , African Swine Fever/virology , African Swine Fever Virus/chemistry , African Swine Fever Virus/genetics , African Swine Fever Virus/immunology , African Swine Fever Virus/pathogenicity , Interferon Type I/antagonists & inhibitors , Interferon Type I/immunology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Nucleotides, Cyclic/antagonists & inhibitors , Nucleotides, Cyclic/metabolism , Swine/immunology , Swine/virology , Vaccines, Attenuated/immunology , Viral Proteins/metabolism , Viral Vaccines/immunology , Host Microbial InteractionsABSTRACT
IMPORTANCE: African swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007 through Russia, Eastern Europe, and more recently to Western European countries, and since 2018 emerged in China, from where it spread throughout Southeast Asia. Recently, outbreaks have appeared in the Caribbean, threatening the Americas. It is estimated that more than 900,000 animals have died directly or indirectly from ASFV since 2021 alone. One of the features of ASFV infection is hemoadsorption (HAD), which has been linked to virulence, although the molecular and pathological basis of this hypothesis remains largely unknown. In this study, we have analyzed and identified the key players responsible of HAD, contributing to the identification of new determinants of ASFV virulence, the understanding of ASFV pathogenesis, and the rational development of new vaccines.
Subject(s)
African Swine Fever Virus , African Swine Fever , Hemadsorption , Protein Sorting Signals , Viral Proteins , Animals , African Swine Fever/virology , African Swine Fever Virus/pathogenicity , Glycosylation , Swine/virology , Virulence , Viral Proteins/chemistry , Viral Proteins/metabolismABSTRACT
IMPORTANCE: Gaining insight into the cell-entry mechanisms of swine acute diarrhea syndrome coronavirus (SADS-CoV) is critical for investigating potential cross-species infections. Here, we demonstrated that pretreatment of host cells with tunicamycin decreased SADS-CoV attachment efficiency, indicating that N-linked glycosylation of host cells was involved in SADS-CoV entry. Common N-linked sugars Neu5Gc and Neu5Ac did not interact with the SADS-CoV S1 protein, suggesting that these molecules were not involved in SADS-CoV entry. Additionally, various host proteases participated in SADS-CoV entry into diverse cells with different efficiencies. Our findings suggested that SADS-CoV may exploit multiple pathways to enter cells, providing insights into intervention strategies targeting the cell entry of this virus.