RESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus, is prevalent in natural reservoir pigs and infects mice. This raises concerns about host jumping or spillover, but little is known about the cause of occurrence. Here, we revealed that dipeptidyl peptidase 4 (DPP4) is a candidate binding target of PHEV spikes and works as a broad barrier to overcome. Investigations of the host breadth of PHEV confirmed that cells derived from pigs and mice are permissive to virus propagation. Both porcine DPP4 and murine DPP4 have high affinity for the viral spike receptor-binding domain (RBD), independent of their catalytic activity. Loss of DPP4 expression results in limited PHEV infection. Structurally, PHEV spike protein binds to the outer surface of blades IV and V of the DPP4 ß-propeller domain, and the DPP4 residues N229 and N321 (relative to human DPP4 numbering) participate in RBD binding via its linked carbohydrate entities. Removal of these N-glycosylations profoundly enhanced the RBD-DPP4 interaction and viral invasion, suggesting they act as shielding in PHEV infection. Furthermore, we found that glycosylation, rather than structural differences or surface charges, is more responsible for DPP4 recognition and species barrier formation. Overall, our findings shed light on virus-receptor interactions and highlight that PHEV tolerance to DPP4 orthologs is a putative determinant of its cross-species transmission or host range expansion.IMPORTANCEPHEV is a neurotropic betacoronavirus that is circulating worldwide and has raised veterinary and economic concerns. In addition to being a reservoir species of pigs, PHEV can also infect wild-type mice, suggesting a "host jump" event. Understanding cross-species transmission is crucial for disease prevention and control but remains to be addressed. Herein, we show that the multifunctional receptor DPP4 plays a pivotal role in the host tropism of PHEV and identifies the conserved glycosylation sites in DPP4 responsible for this restriction. These findings highlight that the ability of PHEV to utilize DPP4 orthologs potentially affects its natural host expansion.
Assuntos
Dipeptidil Peptidase 4 , Especificidade de Hospedeiro , Glicoproteína da Espícula de Coronavírus , Animais , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidase 4/genética , Suínos , Camundongos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Humanos , Ligação Proteica , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/transmissão , Doenças dos Suínos/virologia , Glicosilação , Internalização do Vírus , Linhagem Celular , Betacoronavirus 1/metabolismo , Células HEK293 , DeltacoronavirusRESUMO
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.
Assuntos
Betacoronavirus 1 , Infecções por Coronavirus , Exocitose , Lisossomos , Neurônios , Animais , Camundongos , Betacoronavirus 1/metabolismo , Lisossomos/enzimologia , Lisossomos/metabolismo , Lisossomos/virologia , Vírus da Hepatite Murina/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , SARS-CoV-2/metabolismo , Suínos/virologia , Concentração de Íons de Hidrogênio , ATPases Vacuolares Próton-Translocadoras/metabolismo , Infecções por Coronavirus/patologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologiaRESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic coronavirus belonging to the genus Betacoronavirus. Similar to pathogenic coronaviruses to which humans are susceptible, such as SARS-CoV-2, PHEV is transmitted primarily through respiratory droplets and close contact, entering the central nervous system (CNS) from the peripheral nerves at the site of initial infection. However, the neuroinvasion route of PHEV are poorly understood. Here, we found that BALB/c mice are susceptible to intranasal PHEV infection and showed distinct neurological manifestations. The behavioral study and histopathological examination revealed that PHEV attacks neurons in the CNS and causes significant smell and taste dysfunction in mice. By tracking neuroinvasion, we identified that PHEV invades the CNS via the olfactory nerve and trigeminal nerve located in the nasal cavity, and olfactory sensory neurons (OSNs) were susceptible to viral infection. Immunofluorescence staining and ultrastructural observations revealed that viral materials traveling along axons, suggesting axonal transport may engage in rapid viral transmission in the CNS. Moreover, viral replication in the olfactory system and CNS is associated with inflammatory and immune responses, tissue disorganization and dysfunction. Overall, we proposed that PHEV may serve as a potential prototype for elucidating the pathogenesis of coronavirus-associated neurological complications and olfactory and taste disorders.
Assuntos
Betacoronavirus 1 , COVID-19 , Infecções por Coronavirus/patologia , Transtornos do Olfato , Animais , Betacoronavirus 1/fisiologia , Humanos , Camundongos , Transtornos do Olfato/virologia , SARS-CoV-2 , Olfato , SuínosRESUMO
The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrate that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2α phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2α pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2α also promoted the formation of stress granules (SGs), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR, and eIF2α) for antiviral drugs. IMPORTANCE Coronavirus diseases are caused by different coronaviruses of importance in humans and animals, and specific treatments are extremely limited. ERS, which can activate the UPR to modulate viral replication and the host innate response, is a frequent occurrence in coronavirus-infected cells. PHEV, a neurotropic betacoronavirus, causes nerve cell damage, which accounts for the high mortality rates in suckling piglets. However, it remains incompletely understood whether the highly developed ER in nerve cells plays an antiviral role in ERS and how ERS regulates viral proliferation. In this study, we found that PHEV infection induced ERS and activated the UPR both in vitro and in vivo and that the activated PERK/PKR-eIF2α axis inhibited PHEV replication through attenuating global protein translation and promoting SG formation. A better understanding of coronavirus-induced ERS and UPR activation may reveal the pathogenic mechanism of coronavirus and facilitate the development of new treatment strategies for these diseases.
Assuntos
Betacoronavirus 1/fisiologia , Infecções por Coronavirus/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Grânulos de Estresse/metabolismo , Replicação Viral/fisiologia , eIF-2 Quinase/metabolismo , Animais , Betacoronavirus 1/metabolismo , Linhagem Celular , Infecções por Coronavirus/virologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático , Camundongos , Fosforilação , Biossíntese de Proteínas , Transdução de Sinais , Resposta a Proteínas não DobradasRESUMO
Orf virus (ORFV) is the causative agent of contagious ecthyma, which is an important zoonotic pathogen with a widespread distribution affecting sheep, goats and humans. Our previous research showed that autophagy can be induced in host cells by ORFV infection. However, the exact mechanism of ORFV-induced autophagy remains unknown. In this study, we investigated the underlying mechanisms of autophagy induced by ORFV in OFTu cells and the impact of autophagy on ORFV replication. By using specific autophagy inhibitors and activators, Western blotting, immunofluorescence and transmission electron microscopy imaging, we confirmed that ORFV infection triggered intracellular autophagosome accumulation and the activation of autophagic flux. Moreover, ORFV-induced autophagic activity was found to rely on an increase in the phosphorylation of tuberous sclerosis complex 2 (TSC2) and a decrease in the phosphorylation of mammalian target of rapamycin (mTOR), which is mediated by the suppression of the PI3K/AKT/mTOR signalling pathway and activation of the ERK1/2/mTOR signalling pathway. Furthermore, we investigated the role of mTOR-mediated autophagy during ORFV replication using pharmacological agents and demonstrated that ORFV-induced autophagy correlated positively with viral replication. Taken together, our data reveal the pathways of ORFV-induced autophagy and the impact of autophagy on ORFV replication, providing new insights into ORFV pathogenesis.
Assuntos
Vírus do Orf , Animais , Humanos , Autofagia , Sistema de Sinalização das MAP Quinases , Vírus do Orf/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ovinos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Replicação ViralRESUMO
Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (ß-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing ULK1-knockout neuroblastoma cells, we have identified that ULK1 is not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of noncanonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic ß-CoV and the host. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic alongside the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) pose Betacoronavirus (ß-CoV) as a global public health challenge. Coronaviruses subvert, hijack, or utilize autophagy to promote proliferation, and thus, exploring the cross talk between ß-CoV and autophagy is of great significance in confronting future ß-CoV outbreaks. Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic ß-CoV that invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction is yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypasses the multifaceted regulation of ULK1 kinase. The PHEV-triggered noncanonical autophagy underscores the complex interactions of virus and host and will help in the development of therapeutic strategies targeting noncanonical autophagy to treat ß-CoV disease.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Betacoronavirus 1/metabolismo , Animais , Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , COVID-19 , Linhagem Celular , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/metabolismo , Fosforilação , SARS-CoV-2RESUMO
Orf virus (ORFV) is a highly epitheliotropic parapoxvirus with zoonotic significance that induces proliferative lesions in the skin of sheep, goats, and humans. Several viral proteins carried by ORFV, including nuclear factor-κB (NF-κB) inhibitors, play important roles in hijacking host-associated proteins for viral evasion of the host innate immune response. However, the roles of proteins with unknown functions in viral replication and latent infection remain to be explored. Here, we present data demonstrating that the ORF120, an early-late ORFV-encoded protein, activates the NF-κB pathway in the early phase of infection, which implies that ORFV may regulate NF-κB through a biphasic mechanism. A DUAL membrane yeast two-hybrid system and coimmunoprecipitation experiments revealed that the ORF120 protein interacts with Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1). The overexpression of the ORF120 protein can efficiently increase the expression of G3BP1 and nuclear translocation of NF-κB-p65 in primary ovine fetal turbinate (OFTu) and HeLa cells. The knockdown of G3BP1 significantly decreased ORF120-induced NF-κB activation, indicating that G3BP1 is involved in ORF120-induced NF-κB pathway activation. A dual-luciferase reporter assay revealed that ORF120 could positively regulate the NF-κB pathway through the full-length G3BP1 or the domain of G3BP1RRM+RGG. In conclusion, we demonstrate, for the first time, that the ORF120 protein is capable of positively regulating NF-κB signaling by interacting with G3BP1, providing new insights into ORFV pathogenesis and a theoretical basis for antiviral drug design. IMPORTANCE As part of the host innate response, the nuclear factor-κB (NF-κB) pathway plays a partial antiviral role in nature by regulating the innate immune response. Thus, the NF-κB pathway is probably the most frequently targeted intracellular pathway for subversion by anti-immune modulators that are carried by a wide range of pathogens. Various viruses, including poxviruses, carry several proteins that prepare the host cell for viral replication by inhibiting cytoplasmic events, leading to the initiation of NF-κB transcriptional activity. However, NF-κB activity is hypothesized to facilitate viral replication to a great extent. The significance of our research is in the exploration of the activation mechanism of NF-κB induced by the Orf virus (ORFV) ORF120 protein interacting with G3BP1, which helps not only to explain the ability of ORFV to modulate the immune response through the positive regulation of NF-κB but also to show the mechanism by which the virus evades the host innate immune response.
Assuntos
DNA Helicases/metabolismo , Ectima Contagioso/virologia , NF-kappa B/metabolismo , Vírus do Orf/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas Virais/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , DNA Helicases/química , Células HeLa , Humanos , Vírus do Orf/genética , Vírus do Orf/crescimento & desenvolvimento , Vírus do Orf/patogenicidade , Proteínas de Ligação a Poli-ADP-Ribose/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , Proteínas com Motivo de Reconhecimento de RNA/química , Ovinos , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Ativação Transcricional , Proteínas Virais/genética , VirulênciaRESUMO
BACKGROUND: Porcine hemagglutinating encephalomyelitis virus (PHEV), a member of the genus Betacoronavirus, is the causative agent of neurological disease in pigs. No effective therapeutics are currently available for PHEV infection. Resveratrol has been shown to exert neuroprotective and antiviral effects. Here resveratrol was investigated for its ability to inhibit PHEV replication in nerve cells and central nervous system tissues. METHODS: Anti-PHEV effect of resveratrol was evaluated using an in vitro cell-based PHEV infection model and employing a mouse PHEV infection model. The collected cells or tissues were used for quantitative PCR analysis, western blot analysis, or indirect immunofluorescence assay. The supernatants were collected to quantify viral loads by TCID50 assay in vitro. EC50 and CC50 were determined by dose-response experiments, and the ratio (EC50/CC50) was used as a selectivity index (SI) to measure the antiviral versus cytotoxic activity. RESULTS: Our results showed that resveratrol treatment reduced PHEV titer in a dose-dependent manner, with a 50% inhibition concentration of 6.24 µM. A reduction of > 70% of viral protein expression and mRNA copy number and a 19-fold reduction of virus titer were achieved when infected cells were treated with 10 µM resveratrol in a pre-treatment assay. Quantitative PCR analysis and TCID50 assay results revealed that the addition of 10 µM resveratrol to cells after adsorption of PHEV significantly reduced 56% PHEV mRNA copy number and eightfold virus titer. 10 µM resveratrol treatment reduced 46% PHEV mRNA copy number and fourfold virus titer in virus inactivation assay. Moreover, the in vivo data obtained in this work also demonstrated that resveratrol inhibited PHEV replication, and anti-PHEV activities of resveratrol treatment via intranasal installation displayed better than oral gavage. CONCLUSION: These results indicated that resveratrol exerted antiviral effects under various drug treatment and virus infection conditions in vitro and holds promise as a treatment for PHEV infection in vivo.
Assuntos
Betacoronavirus 1 , Camundongos , Suínos , Animais , Resveratrol/farmacologia , Resveratrol/metabolismo , Betacoronavirus 1/genética , Betacoronavirus 1/metabolismo , Neurônios , Antivirais/farmacologia , Antivirais/metabolismo , Replicação ViralRESUMO
The recent novel coronavirus (SARS-CoV-2) disease (COVID-19) outbreak created a severe public health burden worldwide. Unfortunately, the SARS-CoV-2 variant is still spreading at an unprecedented speed in many countries and regions. There is still a lack of effective treatment for moderate and severe COVID-19 patients, due to a lack of understanding of the SARS-CoV-2 life cycle. Lysosomes, which act as "garbage disposals" for nearly all types of eukaryotic cells, were shown in numerous studies to support SARS-CoV-2 replication. Lysosome-associated pathways are required for virus entry and exit during replication. In this review, we summarize experimental evidence demonstrating a correlation between lysosomal function and SARS-CoV-2 replication, and the development of lysosomal perturbation drugs as anti-SARS-CoV-2 agents.
Assuntos
Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Antivirais/uso terapêutico , Humanos , Lisossomos/metabolismo , Replicação ViralRESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic virus that causes diffuse neuronal infection with neurological damage and high mortality. Virus-induced cytoskeletal dynamics are thought to be closely related to this type of nerve damage. Currently, the regulation pattern of the actin cytoskeleton and its molecular mechanism remain unclear when PHEV enters the host cells. Here, we demonstrate that entry of PHEV into N2a cells induces a biphasic remodeling of the actin cytoskeleton and a dynamic change in cofilin activity. Viral entry is affected by the disruption of actin kinetics or alteration of cofilin activity. PHEV binds to integrin α5ß1 and then initiates the integrin α5ß1-FAK signaling pathway, leading to virus-induced early cofilin phosphorylation and F-actin polymerization. Additionally, Ras-related C3 botulinum toxin substrate 1 (Rac1), cell division cycle 42 (Cdc42), and downstream regulatory gene p21-activated protein kinases (PAKs) are recruited as downstream mediators of PHEV-induced dynamic changes of the cofilin activity pathway. In conclusion, we demonstrate that PHEV utilizes the integrin α5ß1-FAK-Rac1/Cdc42-PAK-LIMK-cofilin pathway to cause an actin cytoskeletal rearrangement to promote its own invasion, providing theoretical support for the development of PHEV pathogenic mechanisms and new antiviral targets.IMPORTANCE PHEV, a member of the Coronaviridae family, is a typical neurotropic virus that primarily affects the nervous system of piglets to produce typical neurological symptoms. However, the mechanism of nerve damage caused by the virus has not been fully elucidated. Actin is an important component of the cytoskeleton of eukaryotic cells and serves as the first obstacle to the entry of pathogens into host cells. Additionally, the morphological structure and function of nerve cells depend on the dynamic regulation of the actin skeleton. Therefore, exploring the mechanism of neuronal injury induced by PHEV from the perspective of the actin cytoskeleton not only helps elucidate the pathogenesis of PHEV but also provides a theoretical basis for the search for new antiviral targets. This is the first report to define a mechanistic link between alterations in signaling from cytoskeleton pathways and the mechanism of PHEV invading nerve cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Betacoronavirus 1/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Integrina alfa5beta1/metabolismo , Degeneração Neural/veterinária , Animais , Linhagem Celular , Infecções por Coronavirus/patologia , Degeneração Neural/virologia , Suínos , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a typical neurotropic coronavirus that mainly invades the central nervous system (CNS) in piglets and causes vomiting and wasting disease. Emerging evidence suggests that PHEV alters microRNA (miRNA) expression profiles, and miRNA has also been postulated to be involved in its pathogenesis, but the mechanisms underlying this process have not been fully explored. In this study, we found that PHEV infection upregulates miR-142a-3p RNA expression in N2a cells and in the CNS of mice. Downregulation of miR-142a-3p by an miRNA inhibitor led to a significant repression of viral proliferation, implying that it acts as a positive regulator of PHEV proliferation. Using a dual-luciferase reporter assay, miR-142a-3p was found to bind directly bound to the 3' untranslated region (3'UTR) of Rab3a mRNA and downregulate its expression. Knockdown of Rab3a expression by transfection with an miR-142a-3p mimic or Rab3a siRNA significantly increased PHEV replication in N2a cells. Conversely, the use of an miR-142a-3p inhibitor or overexpression of Rab3a resulted in a marked restriction of viral production at both the mRNA and protein level. Our data demonstrate that miR-142a-3p promotes PHEV proliferation by directly targeting Rab3a mRNA, and this provides new insights into the mechanisms of PHEV-related pathogenesis and virus-host interactions.
Assuntos
Betacoronavirus 1/genética , Proliferação de Células/genética , Infecções por Coronavirus/genética , MicroRNAs/genética , Suínos/virologia , Proteína rab3A de Ligação ao GTP/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Regulação para Baixo/genética , Células HEK293 , Humanos , Camundongos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Regulação para Cima/genéticaRESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus and causes neurological dysfunction in the central nervous system (CNS), but the neuropathological mechanism of PHEV remains poorly understood. We report that Unc51-like kinase 1 (Ulk1/Unc51.1) is a pivotal regulator of PHEV-induced neurological disorders and functions to selectively control the initiation of nerve growth factor (NGF)/TrkA endosome trafficking. We first identified the function of Ulk1 by histopathologic evaluation in a PHEV-infected mouse model in which neuronal loss was accompanied by the suppression of Ulk1 expression. Morphogenesis assessments in the primary cortical neurons revealed that overexpression or mutations of Ulk1 modulated neurite outgrowth, collateral sprouting, and endosomal transport. Likewise, Ulk1 expression was decreased following PHEV infection, suggesting that there was a correlation between the neurodegeneration and functional Ulk1 deficiency. We then showed that Ulk1 forms a multiprotein complex with TrkA and the early endosome marker Rab5 and that Ulk1 defects lead to either blocking of NGF/TrkA endocytosis or premature degradation of pTrkA via constitutive activation of the Rab5 GTPase. Further investigation determined that the ectopic expression of Rab5 mutants induces aberrant endosomal accumulation of activated pTrkA, proving that targeting of Ulk1-TrkA-NGF signaling to the retrograde transport route in the neurodegenerative process that underlies PHEV infection is dependent on Rab5 GTPase activity. Therefore, we described a long-distance signaling mechanism of PHEV-driven deficits in neurons and suggested that such Ulk1 repression may result in limited NGF/TrkA retrograde signaling within activated Rab5 endosomes, explaining the progressive failure of neurite outgrowth and survival.IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV) is a neurotropic coronavirus and targets neurons in the nervous system for proliferation, frequently leaving behind grievous neurodegeneration. Structural plasticity disorders occur in the axons, dendrites, and dendritic spines of PHEV-infected neurons, and dysfunction of this neural process may contribute to neurologic pathologies, but the mechanisms remain undetermined. Further understanding of the neurological manifestations underlying PHEV infection in the CNS may provide insights into both neurodevelopmental and neurodegenerative diseases that may be conducive to targeted approaches for treatment. The significance of our research is in identifying an Ulk1-related neurodegenerative mechanism, focusing on the regulatory functions of Ulk1 in the transport of long-distance trophic signaling endosomes, thereby explaining the progressive failure of neurite outgrowth and survival associated with PHEV aggression. This is the first report to define a mechanistic link between alterations in signaling from endocytic pathways and the neuropathogenesis of PHEV-induced CNS disease.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Betacoronavirus 1/crescimento & desenvolvimento , Infecções por Coronavirus/veterinária , Fator de Crescimento Neural/metabolismo , Doenças Neurodegenerativas/veterinária , Receptor trkA/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Camundongos , Doenças Neurodegenerativas/patologia , Transdução de SinaisRESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus that invades the central nervous system (CNS) in piglets. Although important progress has been made toward understanding the biology of PHEV, many aspects of its life cycle remain obscure. Here we dissected the molecular mechanism underlying cellular entry and intracellular trafficking of PHEV in mouse neuroblastoma (Neuro-2a) cells. We first performed a thin-section transmission electron microscopy (TEM) assay to characterize the kinetics of PHEV, and we found that viral entry and transfer occur via membranous coating-mediated endo- and exocytosis. To verify the roles of distinct endocytic pathways, systematic approaches were used, including pharmacological inhibition, RNA interference, confocal microscopy analysis, use of fluorescently labeled virus particles, and overexpression of a dominant negative (DN) mutant. Quantification of infected cells showed that PHEV enters cells by clathrin-mediated endocytosis (CME) and that low pH, dynamin, cholesterol, and Eps15 are indispensably involved in this process. Intriguingly, PHEV invasion leads to rapid actin rearrangement, suggesting that the intactness and dynamics of the actin cytoskeleton are positively correlated with viral endocytosis. We next investigated the trafficking of internalized PHEV and found that Rab5- and Rab7-dependent pathways are required for the initiation of a productive infection. Furthermore, a GTPase activation assay suggested that endogenous Rab5 is activated by PHEV and is crucial for viral progression. Our findings demonstrate that PHEV hijacks the CME and endosomal system of the host to enter and traffic within neural cells, providing new insights into PHEV pathogenesis and guidance for antiviral drug design.IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a nonsegmented, positive-sense, single-stranded RNA coronavirus, invades the central nervous system (CNS) and causes neurological dysfunction. Neural cells are its targets for viral progression. However, the detailed mechanism underlying PHEV entry and trafficking remains unknown. PHEV is the etiological agent of porcine hemagglutinating encephalomyelitis, which is an acute and highly contagious disease that causes numerous deaths in suckling piglets and enormous economic losses in China. Understanding the viral entry pathway will not only advance our knowledge of PHEV infection and pathogenesis but also open new approaches to the development of novel therapeutic strategies. Therefore, we employed systematic approaches to dissect the internalization and intracellular trafficking mechanism of PHEV in Neuro-2a cells. This is the first report to describe the process of PHEV entry into nerve cells via clathrin-mediated endocytosis in a dynamin-, cholesterol-, and pH-dependent manner that requires Rab5 and Rab7.
Assuntos
Betacoronavirus 1/fisiologia , Colesterol/metabolismo , Clatrina/metabolismo , Endocitose , Internalização do Vírus , Proteínas rab5 de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Betacoronavirus 1/efeitos dos fármacos , Betacoronavirus 1/genética , Betacoronavirus 1/patogenicidade , Linhagem Celular Tumoral , Dinaminas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Camundongos , Mutação , Neuroblastoma , Interferência de RNARESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus betacoronavirus within the family coronaviridae, which invades the central nervous system (CNS) via peripheral nervous system and causes encephalomyelitis or vomiting and wasting disease (VWD) in sucking piglets. Up to now, although few complete nucleotide sequences of PHEV have been reported, they are not annotated. This study aimed to illuminate genome characterization, phylogenesis and pathogenicity of the PHEV/2008 strain. The full length of the PHEV/2008 strain genome was 30,684 bp, with a G + C content of 37.27%. The genome included at a minimum of 11 predicted open reading frames (ORFs) flanked by 5' and 3' untranslated regions (UTR) of 211 and 289 nucleotides. The replicase polyproteins pp1a and pp1ab, which had 4382 and 7094 amino acid residues, respectively, were predicted to be cleaved into 16 subunits by two viral proteinases. Phylogenetic analysis based on the complete genome sequence revealed that PHEV/2008 strain was genetically different from other known PHEV types, which represented a novel genotype (GI-1). In addition, we found that PHEV/2008 was neurotropic and highly pathogenic to 4-week-old BALB/c mice. Taken together, this is the first detailed annotated, complete genomic sequence of a new genotype PHEV strain in China.
Assuntos
Betacoronavirus 1/genética , Betacoronavirus 1/patogenicidade , Genoma Viral , Animais , Betacoronavirus 1/isolamento & purificação , China , Clonagem Molecular , Infecções por Coronavirus/virologia , DNA Viral , Feminino , Humanos , Camundongos Endogâmicos BALB C , Tipagem Molecular , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Suínos/virologia , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
OBJECTIVE: The spike (S) protein of porcine hemagglutinating encephalomyelitis virus (PHEV) may mediate infection by binding to a cellular neural cell adhesion molecule (NCAM). This study aimed to identify the crucial domain of the S1 subunit of the S protein that interacts with NCAM. METHODS: Three truncated segments (S(1-291), S(277-794) and S(548-868)) of the S gene of PHEV and the NCAM gene were cloned individually into the Escherichia coli expression vectors and yeast two-hybrid expression vectors. The interaction between S(1-291), S(277-794), S(548-868) and NCAM were detected by a GST pull-down experiment and yeast two-hybrid assay. RESULTS: Three fusion proteins (S(1-291), S(277-794) and S(548-868)) were screened for their interactions with NCAM by protein-protein interaction assays. The results of these assays clarified that S(277-794) interacted with NCAM, while S(1-291) and S(548-868) did not. CONCLUSIONS: A small fragment (258-amino-acid fragment, residues 291-548) on the PHEV S protein was posited to be the minimum number of amino acids necessary to interact with NCAM. This fragment may be the receptor-binding domain that mediates PHEV binding to NCAM.
Assuntos
Coronavirus/química , Coronavirus/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Aminoácidos , Coronavirus/genética , Escherichia coli/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Porcine haemagglutinating encephalomyelitis virus (PHEV) is the main causative agent of porcine coronavirus-associated disease, which is characterized by encephalomyelitis and involves the central nervous system. Little is known about the molecular mechanisms of brain injury caused by PHEV. To gain insight into the interaction between the virus and host cells, changes in global gene expression in the cerebral cortex of PHEV- or mock-infected mice were investigated using DNA microarray analysis and quantitative real-time PCR. The results of the microarray analysis showed that 365 genes on day 3 post-infection (p.i.) and 781 genes on day 5 p.i. were differentially expressed in response to PHEV infection in the cerebral cortex. The upregulated genes were mainly involved in immune system processes, antigen processing and presentation, the Jak-STAT signalling pathway, the RIG-I-like receptor signalling pathway, Toll-like receptor signalling and apoptosis-related proteases. Significantly downregulated genes were mainly involved in nervous-system development, synaptic transmission, neuron-projection development, the transmission of nerve impulses and negative regulation of glial cell differentiation. The differential expression of these genes suggests a strong antiviral host response, but may also contribute to the pathogenesis of PHEV resulting in encephalomyelitis.
Assuntos
Córtex Cerebral/patologia , Córtex Cerebral/virologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Expressão Gênica , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In this study, we investigated an acute outbreak of porcine hemagglutinating encephalomyelitis on a farm of 127 pigs in Jilin province, China. Porcine hemagglutinating encephalomyelitis virus (PHEV) was detected in suckling and weaning pigs by RT-PCR assays. Coronavirus-like particles were observed by electron microscopy. The virus isolate was designated PHEV-JT06. The clinical signs, nervous symptoms and positive labeling of neurons in the cerebral cortex with an immunohistochemical stain in PHEV-JT06-infected BALB/c mice supported the diagnosis of PHEV infection. The five full-length PHEV-JT06 structural genes were cloned, sequenced and analyzed. Phylogenetic studies based on the nucleotide and amino acid sequences of the five genes in the outbreak showed that PHEV remained genetically stable. PHEV shares 95.3-99.3% amino acid sequence identity with American strains (AY078417), suggesting that the Chinese isolate is most likely derived from the North American strain. Additionally, PHEV, HCoV-OC43 and BCoV were genetically close. These results may provide some insights into the genotype of the etiological agent responsible for the porcine hemagglutinating encephalomyelitis outbreak and could also provide a comparative view of the genomics of the five structural proteins of PHEV.
Assuntos
Infecções por Coronavirus/epidemiologia , Coronavirus/isolamento & purificação , Surtos de Doenças , Doenças dos Suínos/epidemiologia , Animais , Animais Recém-Nascidos , China/epidemiologia , Clonagem Molecular , Análise por Conglomerados , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Feminino , Variação Genética , Histocitoquímica , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sus scrofa , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Proteínas Virais/genéticaRESUMO
Porcine idiopathic vesicular disease (PIVD), one of several clinically indistinguishable vesicular diseases of pigs, is caused by the emerging pathogen Senecavirus A (SVA). Despite the widespread prevalence of porcine SVA infection, no effective commercial vaccines for PIVD prevention and control are available, due to high costs associated with vaccine testing in pigs, considerable SVA diversity, and SVA rapid evolution. In this study, SVA CH/JL/2022 (OP562896), a novel mutant SVA strain derived from an isolate obtained from a pig farm in Jilin Province, China, was inactivated then combined with four adjuvants, MONTANIDETM GEL02 PR (GEL 02), MONTANIDETM ISA 201 VG (ISA 201), MONTANIDETM IMG 1313 VG N (IMS1313), or Rehydragel LV (LV). The resulting inactivated SVA CH/JL/2022 vaccines were assessed for efficacy in mice and found to induce robust in vivo lymphocyte proliferation responses and strong IgG1, IgG2a, and neutralizing antibody responses with IgG2a/IgG1 ratios of <1. Furthermore, all vaccinated groups exhibited significantly higher levels of serum cytokines IL-2, IL-4, IL-6, and IFN as compared to unvaccinated mice. These results indicate that all vaccines elicited both Th1 and Th2 responses, with Th2 responses predominating. Moreover, vaccinated mice exhibited enhanced resistance to SVA infection, as evidenced by reduced viral RNA levels and SVA infection-induced histopathological changes. Collectively, our results demonstrate that the SVA-GEL vaccine induced more robust immunological responses in mice than did the other three vaccines, thus highlighting the potential of SVA-GEL to serve an effective tool for preventing and controlling SVA infection.
RESUMO
Orf virus (ORFV), a typical member of the genus Parapoxvirus, Poxvirus family, causes a contagious pustular dermatitis in sheep, goats, and humans. Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm, which is a viral factor essential to poxvirus replication. Due to its vital role in viral life, vRNAP has emerged as one of the potential drug targets. In the present study, we investigated the antiviral effect of genistein against ORFV infection. We provided evidence that genistein exerted antiviral effect through blocking viral genome DNA transcription/replication and viral protein synthesis and reducing viral progeny, which were dosedependently decreased in genistein-treated cells. Furthermore, we identified that genistein interacted with the vRNAP RPO30 protein by CETSA, molecular modeling and Fluorescence quenching, a novel antiviral target for ORFV. By blocking vRNAP RPO30 protein using antibody against RPO30, we confirmed that the inhibitory effect exerted by genistein against ORFV infection is mediated through the interaction with RPO30. In conclusion, we demonstrate that genistein effectively inhibits ORFV transcription in host cells by targeting vRNAP RPO30, which might be a promising drug candidate against poxvirus infection.
RESUMO
Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the family Coronaviridae, genus Betacoronavirus, and subgenus Embecovirus that causes neurological disorders, vomiting and wasting disease (VWD), or influenza-like illness (ILI) in pigs. Exosomes regulate nearby or distant cells as a means of intercellular communication; however, whether they are involved in the transmission of viral reference materials during PHEV infection is unknown. Here, we collected exosomes derived from PHEV-infected neural cells (PHEV-exos) and validated their morphological, structural, and content characteristics. High-resolution mass spectrometry indicated that PHEV-exos carry a variety of cargoes, including host innate immunity sensors and viral ingredients. Furthermore, transwell analysis revealed that viral ingredients, such as proteins and RNA fragments, could be encapsulated in the exosomes of multivesicular bodies (MVBs) to nonpermissive microglia. Inhibition of exosome secretion could suppress PHEV infection. Therefore, we concluded that the mode of infectious transmission of PHEV is likely through a mixture of virus-modified exosomes and virions and that exosomal export acts as a host strategy to induce an innate response in replicating nonpermissive bystander cells free of immune system recognition. IMPORTANCE The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a large number of deaths worldwide. Clinical neurological complications have occurred in some cases; however, knowledge of the natural history of coronavirus in the central nervous system (CNS) is thus far limited. PHEV is a typical neurotropic betacoronavirus (ß-CoV) that propagates via neural circuits in the host CNS after peripheral incubation rather than through the bloodstream. It is therefore a good prototype pathogen to investigate the neuropathological pathogenesis of acute human coronavirus infection. In this study, we demonstrate a new association between host vesicle-based secretion and PHEV infection, showing that multivesicular-derived exosomes are one of the modes of infectious transmission and that they mediate the transfer of immunostimulatory cargo to uninfected neuroimmune cells. These findings provide novel insights into the treatment and monitoring of neurological consequences associated with ß-CoV, similar to those associated with SARS-CoV-2.