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1.
J Virol ; 98(9): e0068024, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39158347

RESUMO

Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.


Assuntos
Vírus da Hepatite Murina , Proteínas Virais , Replicação Viral , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/patogenicidade , Vírus da Hepatite Murina/imunologia , Vírus da Hepatite Murina/fisiologia , Animais , Camundongos , Virulência , Proteínas Virais/metabolismo , Proteínas Virais/genética , Vírion/metabolismo , Imunidade Inata , Infecções por Coronavirus/virologia , Infecções por Coronavirus/imunologia , Linhagem Celular , Fases de Leitura Aberta , Humanos
2.
J Neuroophthalmol ; 44(3): 319-329, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39164897

RESUMO

BACKGROUND: Optic neuritis (ON), one of the clinical manifestations of the human neurological disease multiple sclerosis (MS), was also reported in patients with COVID-19 infection, highlighting one potential neurological manifestation of SARS-CoV-2. However, the mechanism of ON in these patients is poorly understood. EVIDENCE ACQUISITION: Insight may be gained by studying the neurotropic mouse hepatitis virus (MHV-A59), a ß-coronavirus that belongs to the same family as SARS-CoV-2. RESULTS: Mouse hepatitis virus-A59, or its isogenic spike protein recombinant strains, inoculation in mice provides an important experimental model to understand underpinning mechanisms of neuroinflammatory demyelination in association with acute stage optic nerve inflammation and chronic stage optic nerve demyelination concurrent with axonal loss. Spike is a surface protein that mediates viral binding and entry into host cells, as well as cell-cell fusion and viral spread. Studies have implicated spike-mediated mechanisms of virus-induced neuroinflammatory demyelination by comparing naturally occurring demyelinating (DM) and nondemyelinating (NDM) MHV strains. CONCLUSIONS: Here, we summarize findings in MHV-induced experimental ON and myelitis, using natural DM and NDM strains as well as engineered recombinant strains of MHV to understand the role of spike protein in inducing ON and demyelinating disease pathology. Potential parallels in human coronavirus-mediated ON and demyelination, and insight into potential therapeutic strategies, are discussed.


Assuntos
COVID-19 , Modelos Animais de Doenças , Vírus da Hepatite Murina , Neurite Óptica , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Neurite Óptica/virologia , Neurite Óptica/etiologia , Neurite Óptica/metabolismo , Neurite Óptica/fisiopatologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Camundongos , COVID-19/complicações , Vírus da Hepatite Murina/fisiologia , Humanos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/fisiopatologia , Betacoronavirus 1/metabolismo
3.
Virology ; 598: 110165, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39013305

RESUMO

Epidemics caused by pathogenic viruses are a severe threat to public health worldwide. Electromagnetic waves are a type of noncontact and nonionizing radiation technology that has emerged as an effective tool for inactivating bacterial pathogens. In this study, we used a 9.375 GHz electromagnetic wave to study the inactivation effect and mechanism of electromagnetic waves on MHV-A59, a substitute virus for pathogenic human coronavirus, and to evaluate the inactivation efficiency on different surface materials. We showed that 9.375 GHz electromagnetic waves inactivate MHV-A59 by destroying viral particles, envelopes, or genomes. We also found that 9.375 GHz electromagnetic waves can decrease the infectivity of viruses on the surface of inanimate materials such as plastic, glass, cloth, and wood. In conclusion, our results suggested that the 9.375 GHz electromagnetic wave is a promising disinfection technique for preventing the spread and infection of pathogenic viruses.


Assuntos
Radiação Eletromagnética , Inativação de Vírus , Inativação de Vírus/efeitos da radiação , Desinfecção/métodos , Animais , Vírus da Hepatite Murina/efeitos da radiação , Vírus da Hepatite Murina/fisiologia , Humanos , Linhagem Celular , Vírion/efeitos da radiação
4.
Sci Total Environ ; 944: 173877, 2024 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-38871327

RESUMO

Wastewater-based epidemiology (WBE) has been an important tool for population surveillance during the COVID-19 pandemic and continues to play a key role in monitoring SARS-CoV-2 infection levels following reductions in national clinical testing schemes. Studies measuring decay profiles of SARS-CoV-2 in wastewater have underscored the value of WBE, however investigations have been hampered by high biosafety requirements for SARS-CoV-2 infection studies. Therefore, surrogate viruses with lower biosafety standards have been used for SARS-CoV-2 decay studies, such as murine hepatitis virus (MHV), but few studies have directly compared decay rates of both viruses. We compared the persistence of SARS-CoV-2 and MHV in wastewater, using 50 % tissue culture infectious dose (TCID50) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays to assess infectious virus titre and viral gene markers, respectively. Infectious SARS-CoV-2 and MHV indicate similar endpoints, however observed early decay characteristics differed, with infectious SARS-CoV-2 decaying more rapidly than MHV. We find that MHV is an appropriate infectious virus surrogate for viable SARS-CoV-2, however inconsistencies exist in viral RNA decay parameters, indicating MHV may not be a suitable nucleic acid surrogate across certain temperature regimes. This study highlights the importance of sample preparation and the potential for decay rate overestimation in wastewater surveillance for SARS-CoV-2 and other pathogens.


Assuntos
Vírus da Hepatite Murina , RNA Viral , SARS-CoV-2 , Águas Residuárias , Águas Residuárias/virologia , SARS-CoV-2/genética , Vírus da Hepatite Murina/fisiologia , COVID-19 , Animais , Estabilidade de RNA
5.
J Biol Chem ; 300(4): 107200, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38508315

RESUMO

Interferon (IFN) regulatory factors (IRF) are key transcription factors in cellular antiviral responses. IRF7, a virus-inducible IRF, expressed primarily in myeloid cells, is required for transcriptional induction of interferon α and antiviral genes. IRF7 is activated by virus-induced phosphorylation in the cytoplasm, leading to its translocation to the nucleus for transcriptional activity. Here, we revealed a nontranscriptional activity of IRF7 contributing to its antiviral functions. IRF7 interacted with the pro-inflammatory transcription factor NF-κB-p65 and inhibited the induction of inflammatory target genes. Using knockdown, knockout, and overexpression strategies, we demonstrated that IRF7 inhibited NF-κB-dependent inflammatory target genes, induced by virus infection or toll-like receptor stimulation. A mutant IRF7, defective in transcriptional activity, interacted with NF-κB-p65 and suppressed NF-κB-induced gene expression. A single-action IRF7 mutant, active in anti-inflammatory function, but defective in transcriptional activity, efficiently suppressed Sendai virus and murine hepatitis virus replication. We, therefore, uncovered an anti-inflammatory function for IRF7, independent of transcriptional activity, contributing to the antiviral response of IRF7.


Assuntos
Fator Regulador 7 de Interferon , NF-kappa B , Animais , Humanos , Camundongos , Células HEK293 , Inflamação/genética , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Vírus Sendai/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Replicação Viral , Mutação , Regulação da Expressão Gênica/genética , Vírus da Hepatite Murina/fisiologia , Infecções por Coronavirus/imunologia , Infecções por Respirovirus/imunologia
6.
J Virol ; 98(4): e0017124, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38488361

RESUMO

The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE: In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.


Assuntos
Antivirais , Coronavirus , Vírus da Hepatite Murina , Palmitatos , Animais , Humanos , Camundongos , Antivirais/farmacologia , Antivirais/metabolismo , Cerulenina/metabolismo , Cerulenina/farmacologia , Coronavirus/efeitos dos fármacos , Coronavirus/fisiologia , Gotículas Lipídicas/efeitos dos fármacos , Palmitatos/farmacologia , Ácido Palmítico/farmacologia , Ácido Palmítico/metabolismo , Propiolactona/análogos & derivados , Replicação Viral/efeitos dos fármacos , Vírus da Hepatite Murina/efeitos dos fármacos , Vírus da Hepatite Murina/fisiologia
7.
Viruses ; 16(1)2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38257840

RESUMO

The ongoing COVID-19 pandemic has revealed the shortfalls in our understanding of how to treat coronavirus infections. With almost 7 million case fatalities of COVID-19 globally, the catalog of FDA-approved antiviral therapeutics is limited compared to other medications, such as antibiotics. All-trans retinoic acid (RA), or activated vitamin A, has been studied as a potential therapeutic against coronavirus infection because of its antiviral properties. Due to its impact on different signaling pathways, RA's mechanism of action during coronavirus infection has not been thoroughly described. To determine RA's mechanism of action, we examined its effect against a mouse coronavirus, mouse hepatitis virus strain A59 (MHV). We demonstrated that RA significantly decreased viral titers in infected mouse L929 fibroblasts and RAW 264.7 macrophages. The reduced viral titers were associated with a corresponding decrease in MHV nucleocapsid protein expression. Using interferon regulatory factor 3 (IRF3) knockout RAW 264.7 cells, we demonstrated that RA-induced suppression of MHV required IRF3 activity. RNA-seq analysis of wildtype and IRF3 knockout RAW cells showed that RA upregulated calcium/calmodulin (CaM) signaling proteins, such as CaM kinase kinase 1 (CaMKK1). When treated with a CaMKK inhibitor, RA was unable to upregulate IRF activation during MHV infection. In conclusion, our results demonstrate that RA-induced protection against coronavirus infection depends on IRF3 and CaMKK.


Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Fator Regulador 3 de Interferon , Vírus da Hepatite Murina , Tretinoína , Replicação Viral , Animais , Camundongos , Aminoácidos , Antivirais/farmacologia , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Tretinoína/farmacologia , Replicação Viral/efeitos dos fármacos , Vírus da Hepatite Murina/efeitos dos fármacos , Vírus da Hepatite Murina/fisiologia , Células RAW 264.7 , Células L
8.
J Virol ; 98(1): e0123923, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38099687

RESUMO

Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus-host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus-host interactions and facilitating the development of new broad-spectrum antiviral drugs.IMPORTANCECoronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.


Assuntos
Infecções por Coronavirus , Coronavirus , Quinases Dyrk , Animais , Humanos , Camundongos , Antígenos CD13/genética , Coronavirus/classificação , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Deltacoronavirus , Vírus da Hepatite Murina/fisiologia , Suínos , Vírus da Gastroenterite Transmissível/genética , Tirosina , Replicação Viral/fisiologia , Quinases Dyrk/metabolismo
9.
Viruses ; 15(12)2023 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-38140675

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 (ß-CoV) betacoronavirus has posed a significant threat to global health. Despite the availability of vaccines, the virus continues to spread, and there is a need for alternative strategies to alleviate its impact. Vitamin D, a secosteroid hormone best known for its role in bone health, exhibits immunomodulatory effects in certain viral infections. Here, we have shown that bioactive vitamin D (calcitriol) limits in vitro replication of SARS-CoV-2 and murine coronaviruses MHV-3 and MHV-A59. Comparative studies involving wild-type mice intranasally infected with MHV-3, a model for studying ß-CoV respiratory infections, confirmed the protective effect of vitamin D in vivo. Accordingly, mice fed a standard diet rapidly succumbed to MHV-3 infection, whereas those on a vitamin D-rich diet (10,000 IU of Vitamin D3/kg) displayed increased resistance to acute respiratory damage and systemic complications. Consistent with these findings, the vitamin D-supplemented group exhibited lower viral titers in their lungs and reduced levels of TNF, IL-6, IL-1ß, and IFN-γ, alongside an enhanced type I interferon response. Altogether, our findings suggest vitamin D supplementation ameliorates ß-CoV-triggered respiratory illness and systemic complications in mice, likely via modulation of the host's immune response to the virus.


Assuntos
Vírus da Hepatite Murina , Pneumonia , Camundongos , Humanos , Animais , Vitamina D , Pandemias/prevenção & controle , Vírus da Hepatite Murina/fisiologia , SARS-CoV-2 , Vitaminas/farmacologia , Vitaminas/uso terapêutico , Dieta
10.
J Virol ; 97(8): e0074923, 2023 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-37504572

RESUMO

Interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is critical in restricting neurotropic murine-ß-coronavirus, RSA59 infection. RSA59 intracranial injection of Ifit2-deficient (-/-) compared to wild-type (WT) mice results in impaired acute microglial activation, reduced CX3CR1 expression, limited migration of peripheral lymphocytes into the brain, and impaired virus control followed by severe morbidity and mortality. While the protective role of Ifit2 is established for acute viral encephalitis, less is known about its influence during the chronic demyelinating phase of RSA59 infection. To understand this, RSA59 infected Ifit2-/- and Ifit2+/+ (WT) were observed for neuropathological outcomes at day 5 (acute phase) and 30 post-infection (chronic phase). Our study demonstrates that Ifit2 deficiency causes extensive RSA59 spread throughout the spinal cord gray and white matter, associated with impaired CD4+ T and CD8+ T cell infiltration. Further, the cervical lymph nodes of RSA59 infected Ifit2-/- mice showed reduced activation of CD4+ T cells and impaired IFNγ expression during acute encephalomyelitis. Interestingly, BBB integrity was better preserved in Ifit2-/- mice, as evidenced by tight junction protein Claudin-5 and adapter protein ZO-1 expression surrounding the meninges and blood vessels and decreased Texas red dye uptake, which may be responsible for reduced leukocyte infiltration. In contrast to sparse myelin loss in WT mice, the chronic disease phase in Ifit2-/- mice was associated with severe demyelination and persistent viral load, even at low inoculation doses. Overall, our study highlights that Ifit2 provides antiviral functions by promoting acute neuroinflammation and thereby aiding virus control and limiting severe chronic demyelination. IMPORTANCE Interferons execute their function by inducing specific genes collectively termed as interferon-stimulated genes (ISGs), among which interferon-induced protein with tetratricopeptide repeats 2, Ifit2, is known for restricting neurotropic viral replication and spread. However, little is known about its role in viral spread to the spinal cord and its associated myelin pathology. Toward this, our study using a neurotropic murine ß-coronavirus and Ifit2-deficient mice demonstrates that Ifit2 deficiency causes extensive viral spread throughout the gray and white matter of the spinal cord accompanied by impaired microglial activation and T cell infiltration. Furthermore, infected Ifit2-deficient mice showed impaired activation of T cells in the cervical lymph node and relatively intact blood-brain barrier integrity. Overall, Ifit2 plays a crucial role in mounting host immunity against neurotropic murine coronavirus in the acute phase while preventing mice from developing viral-induced severe chronic neuroinflammatory demyelination, the characteristic feature of human neurological disease multiple sclerosis (MS).


Assuntos
Infecções por Coronavirus , Esclerose Múltipla , Vírus da Hepatite Murina , Substância Branca , Camundongos , Humanos , Animais , Substância Branca/patologia , Vírus da Hepatite Murina/fisiologia , Bainha de Mielina , Interferons , Proteínas/genética , Medula Espinal/patologia , Esclerose Múltipla/patologia , Camundongos Endogâmicos C57BL , Proteínas de Ligação a RNA/genética , Proteínas Reguladoras de Apoptose/genética
11.
Neurochem Int ; 169: 105567, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37348761

RESUMO

COVID-19 affects primarily the lung. However, several other systemic alterations, including muscle weakness, fatigue and myalgia have been reported and may contribute to the disease outcome. We hypothesize that changes in the neuromuscular system may contribute to the latter symptoms observed in COVID-19 patients. Here, we showed that C57BL/6J mice inoculated intranasally with the murine betacoronavirus hepatitis coronavirus 3 (MHV-3), a model for studying COVID-19 in BSL-2 conditions that emulates severe COVID-19, developed robust motor alterations in muscle strength and locomotor activity. The latter changes were accompanied by degeneration and loss of motoneurons that were associated with the presence of virus-like particles inside the motoneuron. At the neuromuscular junction level, there were signs of atrophy and fragmentation in synaptic elements of MHV-3-infected mice. Furthermore, there was muscle atrophy and fiber type switch with alteration in myokines levels in muscles of MHV-3-infected mice. Collectively, our results show that acute infection with a betacoronavirus leads to robust motor impairment accompanied by neuromuscular system alteration.


Assuntos
COVID-19 , Vírus da Hepatite Murina , Camundongos , Animais , Camundongos Endogâmicos C57BL , Neurônios Motores , Junção Neuromuscular , Vírus da Hepatite Murina/fisiologia
12.
J Neuroimmunol ; 381: 578133, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37352687

RESUMO

Intracranial inoculation of susceptible mice with a glial-tropic strain of mouse hepatitis virus (JHMV), a murine coronavirus, results in an acute encephalomyelitis followed by viral persistence in white matter tracts accompanied by chronic neuroinflammation and demyelination. Microglia are the resident immune cell of the central nervous system (CNS) and are considered important in regulating events associated with neuroinflammation as well as influencing both white matter damage and remyelination. To better understand mechanisms by which microglia contribute to these immune-mediated events, JHMV-infected mice with established demyelination were treated with the small molecular inhibitor of colony stimulating factor 1 receptor (CSF1R), PLX5622, to deplete microglia. Treatment with PLX5622 did not affect viral replication within the CNS yet the severity of demyelination was increased and remyelination impaired compared to control mice. Gene expression analysis revealed that targeting microglia resulted in altered expression of genes associated with immune cell activation and phagocytosis of myelin debris. These findings indicate that microglia are not critical in viral surveillance in persistently JHMV-infected mice yet restrict white matter damage and remyelination, in part, by influencing phagocytosis of myelin debris.


Assuntos
Infecções por Coronavirus , Doenças Desmielinizantes , Vírus da Hepatite Murina , Remielinização , Substância Branca , Camundongos , Animais , Microglia/metabolismo , Vírus da Hepatite Murina/fisiologia , Doenças Neuroinflamatórias , Infecções por Coronavirus/complicações , Camundongos Endogâmicos C57BL
13.
Viruses ; 15(1)2023 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-36680255

RESUMO

The ß-Coronavirus mouse hepatitis virus (MHV-A59)-RSA59 has a patent stretch of fusion peptide (FP) containing two consecutive central prolines (PP) in the S2 domain of the Spike protein. Our previous studies compared the PP-containing fusogenic-demyelinating strain RSA59(PP) to its one proline-deleted mutant strain RSA59(P) and one proline-containing non-fusogenic non-demyelinating parental strain RSMHV2(P) to its one proline inserted mutant strain RSMHV2(PP). These studies highlighted the crucial role of PP in fusogenicity, hepato-neuropathogenesis, and demyelination. Computational studies combined with biophysical data indicate that PP at the center of the FP provides local rigidity while imparting global fluctuation to the Spike protein that enhances the fusogenic properties of RSA59(PP) and RSMHV2(PP). To elaborate on the understanding of the role of PP in the FP of MHV, the differential neuroglial tropism of the PP and P mutant strains was investigated. Comparative studies demonstrated that PP significantly enhances the viral tropism for neurons, microglia, and oligodendrocytes. PP, however, is not essential for viral tropism for either astroglial or oligodendroglial precursors or the infection of meningeal fibroblasts in the blood-brain and blood-CSF barriers. PP in the fusion domain is critical for promoting gliopathy, making it a potential region for designing antivirals for neuro-COVID therapy.


Assuntos
Vírus da Hepatite Murina , Glicoproteína da Espícula de Coronavírus , Tropismo Viral , Animais , Camundongos , Vírus da Hepatite Murina/fisiologia , Peptídeos/metabolismo , Prolina , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/metabolismo
14.
J Virol ; 96(16): e0084122, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-35924922

RESUMO

Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.


Assuntos
Exorribonucleases , Aptidão Genética , Vírus da Hepatite Murina , Proteólise , RNA Viral , Proteínas não Estruturais Virais , Proteínas do Complexo da Replicase Viral , Animais , Exorribonucleases/genética , Exorribonucleases/metabolismo , Camundongos , Vírus da Hepatite Murina/enzimologia , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/crescimento & desenvolvimento , Vírus da Hepatite Murina/fisiologia , Mutação , Poliproteínas/química , Poliproteínas/genética , Poliproteínas/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Recombinação Genética , Transcrição Gênica , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas do Complexo da Replicase Viral/química , Proteínas do Complexo da Replicase Viral/genética , Proteínas do Complexo da Replicase Viral/metabolismo , Replicação Viral
15.
Mol Neurobiol ; 59(10): 5970-5986, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35831558

RESUMO

We recently reported acute COVID-19 symptoms, clinical status, weight loss, multi-organ pathological changes, and animal death in a murine hepatitis virus-1 (MHV-1) coronavirus mouse model of COVID-19, which were similar to that observed in humans with COVID-19. We further examined long-term (12 months post-infection) sequelae of COVID-19 in these mice. Congested blood vessels, perivascular cavitation, pericellular halos, vacuolation of neuropils, pyknotic nuclei, acute eosinophilic necrosis, necrotic neurons with fragmented nuclei, and vacuolation were observed in the brain cortex 12 months post-MHV-1 infection. These changes were associated with increased reactive astrocytes and microglia, hyperphosphorylated TDP-43 and tau, and a decrease in synaptic protein synaptophysin-1, suggesting the possible long-term impact of SARS-CoV-2 infection on defective neuronal integrity. The lungs showed severe inflammation, bronchiolar airway wall thickening due to fibrotic remodeling, bronchioles with increased numbers of goblet cells in the epithelial lining, and bronchiole walls with increased numbers of inflammatory cells. Hearts showed severe interstitial edema, vascular congestion and dilation, nucleated red blood cells (RBCs), RBCs infiltrating between degenerative myocardial fibers, inflammatory cells and apoptotic bodies and acute myocyte necrosis, hypertrophy, and fibrosis. Long-term changes in the liver and kidney were less severe than those observed in the acute phase. Noteworthy, the treatment of infected mice with a small molecule synthetic peptide which prevents the binding of spike protein to its respective receptors significantly attenuated disease progression, as well as the pathological changes observed post-long-term infection. Collectively, these findings suggest that COVID-19 may result in long-term, irreversible changes predominantly in the brain, lung, and heart.


Assuntos
COVID-19 , Vírus da Hepatite Murina , Animais , COVID-19/complicações , Progressão da Doença , Humanos , Camundongos , Vírus da Hepatite Murina/fisiologia , Necrose , SARS-CoV-2
16.
Viruses ; 14(2)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35215991

RESUMO

Deletions in the spike gene of mouse hepatitis virus (MHV) produce several variants with diverse biological characteristics, highlighting the significance of the spike gene in viral pathogenesis. In this study, we characterized the JHM-X strain, which has a deletion in the hypervariable region (HVR) of the spike gene, compared with the cl-2 strain, which has a full spike gene. Cytopathic effects (CPEs) induced by the two strains revealed that the size of the CPE produced by cl-2 is much greater than that produced by JHM-X in delayed brain tumor (DBT) cells. Thus, this finding explains the greater fusion activity of cl-2 than JHM-X in cultured cells, and we speculate that the deletion region of the spike protein is involved in the fusion activity differences. In contrast with the fusion activity, a comparison of the virus growth kinetics revealed that the titer of JHM-X was approximately 100 times higher than that of cl-2. We found that the deletion region of the spike protein was involved in fusion activity differences, whereas cl-2 produced significantly higher luciferase activity than JHM-X upon similar expression levels of the spike protein. However, the reason behind the growth difference is still unknown. Overall, we discovered that deletion in the HVR of the spike gene could be involved in the fusion activity differences between the two strains.


Assuntos
Fusão Celular , Vírus da Hepatite Murina/patogenicidade , Glicoproteína da Espícula de Coronavírus/fisiologia , Animais , Linhagem Celular , Camundongos , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/fisiologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/genética
17.
J Virol ; 96(3): e0184221, 2022 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-34817197

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Vírus da Hepatite Murina/fisiologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais Reguladoras e Acessórias/genética , Animais , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Camundongos , Mortalidade , Proteínas Virais Reguladoras e Acessórias/química , Tropismo Viral , Virulência/genética , Fatores de Virulência/genética
19.
Viruses ; 13(9)2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34578284

RESUMO

Infection with SARS-CoV-2, the virus responsible for the global COVID-19 pandemic, causes a respiratory illness that can severely impact other organ systems and is possibly precipitated by cytokine storm, septic shock, thrombosis, and oxidative stress. SARS-CoV-2 infected individuals may be asymptomatic or may experience mild, moderate, or severe symptoms with or without pneumonia. The mechanisms by which SARS-CoV-2 infects humans are largely unknown. Mouse hepatitis virus 1 (MHV-1)-induced infection was used as a highly relevant surrogate animal model for this study. We further characterized this animal model and compared it with SARS-CoV-2 infection in humans. MHV-1 inoculated mice displayed death as well as weight loss, as reported earlier. We showed that MHV-1-infected mice at days 7-8 exhibit severe lung inflammation, peribronchiolar interstitial infiltration, bronchiolar epithelial cell necrosis and intra-alveolar necrotic debris, alveolar exudation (surrounding alveolar walls have capillaries that are dilated and filled with red blood cells), mononuclear cell infiltration, hyaline membrane formation, the presence of hemosiderin-laden macrophages, and interstitial edema. When compared to uninfected mice, the infected mice showed severe liver vascular congestion, luminal thrombosis of portal and sinusoidal vessels, hepatocyte degeneration, cell necrosis, and hemorrhagic changes. Proximal and distal tubular necrosis, hemorrhage in interstitial tissue, and the vacuolation of renal tubules were observed. The heart showed severe interstitial edema, vascular congestion, and dilation, as well as red blood cell extravasation into the interstitium. Upon examination of the MHV-1 infected mice brain, we observed congested blood vessels, perivascular cavitation, cortical pericellular halos, vacuolation of neuropils, darkly stained nuclei, pyknotic nuclei, and associated vacuolation of the neuropil in the cortex, as well as acute eosinophilic necrosis and necrotic neurons with fragmented nuclei and vacuolation in the hippocampus. Our findings suggest that the widespread thrombotic events observed in the surrogate animal model for SARS-CoV-2 mimic the reported findings in SARS-CoV-2 infected humans, representing a highly relevant and safe animal model for the study of the pathophysiologic mechanisms of SARS-CoV-2 for potential therapeutic interventions.


Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Vírus da Hepatite Murina/fisiologia , Animais , Biomarcadores , Biópsia , COVID-19/patologia , COVID-19/virologia , Infecções por Coronavirus/mortalidade , Modelos Animais de Doenças , Feminino , Genoma Viral , Humanos , Imuno-Histoquímica , Testes de Função Hepática , Camundongos , Mortalidade , Especificidade de Órgãos , SARS-CoV-2/fisiologia , Carga Viral
20.
J Virol ; 95(22): e0127621, 2021 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-34495692

RESUMO

The emergence of life-threatening zoonotic diseases caused by betacoronaviruses, including the ongoing coronavirus disease 19 (COVID-19) pandemic, has highlighted the need for developing preclinical models mirroring respiratory and systemic pathophysiological manifestations seen in infected humans. Here, we showed that C57BL/6J wild-type mice intranasally inoculated with the murine betacoronavirus murine hepatitis coronavirus 3 (MHV-3) develop a robust inflammatory response leading to acute lung injuries, including alveolar edema, hemorrhage, and fibrin thrombi. Although such histopathological changes seemed to resolve as the infection advanced, they efficiently impaired respiratory function, as the infected mice displayed restricted lung distention and increased respiratory frequency and ventilation. Following respiratory manifestation, the MHV-3 infection became systemic, and a high virus burden could be detected in multiple organs along with morphological changes. The systemic manifestation of MHV-3 infection was also marked by a sharp drop in the number of circulating platelets and lymphocytes, besides the augmented concentration of the proinflammatory cytokines interleukin 1 beta (IL-1ß), IL-6, IL-12, gamma interferon (IFN-γ), and tumor necrosis factor (TNF), thereby mirroring some clinical features observed in moderate and severe cases of COVID-19. Importantly, both respiratory and systemic changes triggered by MHV-3 infection were greatly prevented by blocking TNF signaling, either via genetic or pharmacologic approaches. In line with this, TNF blockage also diminished the infection-mediated release of proinflammatory cytokines and virus replication of human epithelial lung cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Collectively, results show that MHV-3 respiratory infection leads to a large range of clinical manifestations in mice and may constitute an attractive, lower-cost, biosafety level 2 (BSL2) in vivo platform for evaluating the respiratory and multiorgan involvement of betacoronavirus infections. IMPORTANCE Mouse models have long been used as valuable in vivo platforms to investigate the pathogenesis of viral infections and effective countermeasures. The natural resistance of mice to the novel betacoronavirus SARS-CoV-2, the causative agent of COVID-19, has launched a race toward the characterization of SARS-CoV-2 infection in other animals (e.g., hamsters, cats, ferrets, bats, and monkeys), as well as adaptation of the mouse model, by modifying either the host or the virus. In the present study, we utilized a natural pathogen of mice, MHV, as a prototype to model betacoronavirus-induced acute lung injure and multiorgan involvement under biosafety level 2 conditions. We showed that C57BL/6J mice intranasally inoculated with MHV-3 develops severe disease, which includes acute lung damage and respiratory distress that precede systemic inflammation and death. Accordingly, the proposed animal model may provide a useful tool for studies regarding betacoronavirus respiratory infection and related diseases.


Assuntos
Infecções por Coronavirus/patologia , Modelos Animais de Doenças , Pulmão/patologia , Vírus da Hepatite Murina/patogenicidade , Animais , Linhagem Celular , Contenção de Riscos Biológicos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Humanos , Inflamação , Fígado/patologia , Fígado/virologia , Pulmão/virologia , Camundongos , Vírus da Hepatite Murina/efeitos dos fármacos , Vírus da Hepatite Murina/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Replicação Viral/efeitos dos fármacos
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