Your browser doesn't support javascript.
loading
: 20 | 50 | 100
1 - 7 de 7
1.
J Virol ; 97(12): e0133823, 2023 Dec 21.
Article En | MEDLINE | ID: mdl-38009916

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.


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/virology
2.
mBio ; 14(1): e0305422, 2023 02 28.
Article En | MEDLINE | ID: mdl-36541757

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.


Betacoronavirus 1 , COVID-19 , Exosomes , Swine , Animals , Humans , Betacoronavirus 1/genetics , Betacoronavirus 1/metabolism , SARS-CoV-2
3.
Virol J ; 19(1): 226, 2022 12 28.
Article En | MEDLINE | ID: mdl-36578037

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.


Betacoronavirus 1 , Mice , Swine , Animals , Resveratrol/pharmacology , Resveratrol/metabolism , Betacoronavirus 1/genetics , Betacoronavirus 1/metabolism , Neurons , Antiviral Agents/pharmacology , Antiviral Agents/metabolism , Virus Replication
4.
J Virol ; 96(1): e0169521, 2022 01 12.
Article En | MEDLINE | ID: mdl-34643429

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.


Betacoronavirus 1/physiology , Coronavirus Infections/metabolism , Eukaryotic Initiation Factor-2/metabolism , Stress Granules/metabolism , Virus Replication/physiology , eIF-2 Kinase/metabolism , Animals , Betacoronavirus 1/metabolism , Cell Line , Coronavirus Infections/virology , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Endoplasmic Reticulum Stress , Mice , Phosphorylation , Protein Biosynthesis , Signal Transduction , Unfolded Protein Response
5.
J Virol ; 95(19): e0085121, 2021 09 09.
Article En | MEDLINE | ID: mdl-34287052

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.


Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Autophagy/physiology , Betacoronavirus 1/metabolism , Animals , Autophagosomes/metabolism , Beclin-1/metabolism , COVID-19 , Cell Line , Gene Knockout Techniques , Intracellular Signaling Peptides and Proteins/metabolism , Male , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred BALB C , Neurons/metabolism , Phosphorylation , SARS-CoV-2
6.
J Virol ; 93(5)2019 03 01.
Article En | MEDLINE | ID: mdl-30541856

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.


Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/metabolism , Betacoronavirus 1/metabolism , Focal Adhesion Kinase 1/metabolism , Integrin alpha5beta1/metabolism , Nerve Degeneration/veterinary , Animals , Cell Line , Coronavirus Infections/pathology , Nerve Degeneration/virology , Swine , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases/metabolism
7.
Article En | MEDLINE | ID: mdl-28516065

Porcine hemagglutinating encephalomyelitis virus (PHEV) invades the central nervous system (CNS) and causes neurodegenerative disease in suckling piglets, but the understanding of its neuropathogenicity for neurological dysfunction remains limited. Here, we report that miR-142-5p is localized to neurons and negatively regulates neuronal morphogenesis in porcine hemagglutinating encephalomyelitis (PHE). This phenotype was mediated by miR-142-5p inhibition of an mRNA encoding unc-51-like-kinase1 (Ulk1), which controls axon outgrowth and dendrite formation. Modulating miR-142-5p activity by microRNA mimics or inhibitors induced neurodegeneration, including stunted axon elongation, unstable dendritic spine formation, and irregular swelling and disconnection in neurites. Relieving Ulk1 mRNA repression in primary cortical neurons by miR-142-5p antagomirs or replication-deficient adenoviruses encoding Ulk1 (Ad5-Ulk1), which improved rescue of nerve injury, restricted viral replication, and increased survival rate in mice underlying PHEV infection. In contrast, disrupting Ulk1 in RNAi-expressing neurons mostly led to significantly shortened axon elongation and/or an abnormally large number of branched dendrites. Taken together, we demonstrated that the abnormal neuronal morphogenesis underlying PHEV infection was mainly caused by functional mRNA repression of the miR-142-5p target Ulk1. Our data revealed that PHEV adapted to use spatiotemporal control of host microRNAs to invade CNS, and provided new insights into the virus-associated neurological dysfunction microenvironment.


Autophagy-Related Protein-1 Homolog/drug effects , Autophagy-Related Protein-1 Homolog/metabolism , Betacoronavirus 1/metabolism , MicroRNAs/antagonists & inhibitors , Morphogenesis/drug effects , Neurons/drug effects , Neurons/virology , Animals , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/physiology , Axons/drug effects , Axons/pathology , Axons/virology , Cell Proliferation/drug effects , Cerebral Cortex/drug effects , Cerebral Cortex/pathology , Cerebral Cortex/virology , Coronavirus Infections/virology , Dendrites/drug effects , Dendrites/pathology , Dendrites/virology , Disease Models, Animal , Gene Expression Regulation , Host-Pathogen Interactions , Male , Mice , Mice, Inbred BALB C , MicroRNAs/genetics , MicroRNAs/pharmacology , Neurons/pathology , Rats , Rats, Sprague-Dawley , Virus Replication/drug effects
...