Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Clin Sci (Lond) ; 135(24): 2667-2689, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34807265

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Basigina/metabolismo , Miocárdio/enzimologia , Pericitos/enzimologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/sangue , Células CACO-2 , Morte Celular , Criança , Pré-Escolar , Citocinas/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Miocárdio/citologia , Pericitos/virologia , Cultura Primária de Células , Adulto Jovem
2.
Retrovirology ; 11: 98, 2014 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-25421745

RESUMO

BACKGROUND: HIV-1, like all viruses, is entirely dependent on the host cell for providing the metabolic resources for completion of the viral replication cycle and the production of virions. It is well established that HIV-1 replicates efficiently in activated CD4+ T cells, whereas resting CD4+ T cells are refractory to infection with HIV-1. A hallmark of T cell activation is the upregulation of glycolysis to meet the biosynthetic and bioenergetic needs of cell proliferation and the execution of effector functions by the secretion of cytokines. To date, it has remained unknown if HIV-1 requires the high glycolytic activity of activated T cells to support its replication. RESULTS: We report that in primary CD4+ T cells, the flux through the glycolytic pathway is increased upon infection with HIV-1. This increase in glycolytic activity does not occur in T cell lines when infected with HIV-1. By providing cells with galactose instead of glucose, the former being a poor substrate for glycolysis, we monitored the effect of preventing glycolysis in CD4+ T cells on virus replication cycle and cell fate. We observed that HIV-1 infected primary CD4+ T cells cultured in galactose have a survival advantage over those cultured in glucose and this coincides with reduced caspase 3 activation and apoptosis in cultures with galactose. T cell lines do not recapitulate this difference in cell death. Finally, we demonstrate that virion production is dependent on glycolysis as cultures containing galactose yield reduced amounts of HIV-1 virions compared with cultures containing glucose. CONCLUSIONS: The replication of HIV-1 in primary CD4+ T cells causes an increase in glycolytic flux of the cell. Glycolysis is particularly required for virion production and additionally increases the sensitivity of the infected cell to virus-induced cell death.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Glicólise , HIV-1/fisiologia , Montagem de Vírus , Liberação de Vírus , Replicação Viral , Sobrevivência Celular , Células Cultivadas , Meios de Cultura/química , Metabolismo Energético , Galactose/metabolismo , Glucose/metabolismo , HIV-1/crescimento & desenvolvimento , HIV-1/patogenicidade , Humanos
3.
Viruses ; 16(6)2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38932155

RESUMO

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The coalescence of SARS-CoV-2 with seasonal respiratory viruses, particularly influenza viruses, is a global health concern. To understand this, transgenic mice expressing the human ACE2 receptor (K18-hACE2) were infected with influenza A virus (IAV) followed by SARS-CoV-2 and the host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 alone. The sequentially infected mice showed reduced SARS-CoV-2 RNA synthesis, yet exhibited more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to the singly infected or control mice. Sequential infection also exacerbated the extrapulmonary encephalitic manifestations associated with SARS-CoV-2 infection. Conversely, prior infection with a commercially available, multivalent live-attenuated influenza vaccine (Fluenz Tetra) elicited the same reduction in SARS-CoV-2 RNA synthesis, albeit without the associated increase in disease severity. This suggests that the innate immune response stimulated by IAV inhibits SARS-CoV-2. Interestingly, infection with an attenuated, apathogenic influenza vaccine does not result in an aberrant immune response and enhanced disease severity. Taken together, the data suggest coinfection ('twinfection') is deleterious and mitigation steps should be instituted as part of the comprehensive public health and management strategy of COVID-19.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Modelos Animais de Doenças , Vírus da Influenza A , Camundongos Transgênicos , Infecções por Orthomyxoviridae , SARS-CoV-2 , Animais , COVID-19/imunologia , COVID-19/virologia , Camundongos , SARS-CoV-2/imunologia , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/genética , Humanos , Coinfecção/virologia , Pulmão/virologia , Pulmão/patologia , Encefalite Viral/virologia , Encefalite Viral/imunologia , Vacinas contra Influenza/imunologia , Feminino , Imunidade Inata
4.
Sci Signal ; 15(757): eabm0808, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36282911

RESUMO

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.


Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , SARS-CoV-2/genética , Fosforilação , Quinase 3 da Glicogênio Sintase/metabolismo , Replicação Viral , Proteínas do Nucleocapsídeo/metabolismo , Nucleocapsídeo/metabolismo , Serina/metabolismo , Treonina/metabolismo , Mamíferos/metabolismo , Proteínas Serina-Treonina Quinases
5.
Science ; 370(6517): 725-730, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958580

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.


Assuntos
Ácido Linoleico/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus , Sítios de Ligação , Chlorocebus aethiops , Microscopia Crioeletrônica , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio , Modelos Moleculares , Peptidil Dipeptidase A/metabolismo , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Células Vero
6.
bioRxiv ; 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-32817937

RESUMO

While vaccines are vital for preventing COVID-19 infections, it is critical to develop new therapies to treat patients who become infected. Pharmacological targeting of a host factor required for viral replication can suppress viral spread with a low probability of viral mutation leading to resistance. In particular, host kinases are highly druggable targets and a number of conserved coronavirus proteins, notably the nucleoprotein (N), require phosphorylation for full functionality. In order to understand how targeting kinases could be used to compromise viral replication, we used a combination of phosphoproteomics and bioinformatics as well as genetic and pharmacological kinase inhibition to define the enzymes important for SARS-CoV-2 N protein phosphorylation and viral replication. From these data, we propose a model whereby SRPK1/2 initiates phosphorylation of the N protein, which primes for further phosphorylation by GSK-3a/b and CK1 to achieve extensive phosphorylation of the N protein SR-rich domain. Importantly, we were able to leverage our data to identify an FDA-approved kinase inhibitor, Alectinib, that suppresses N phosphorylation by SRPK1/2 and limits SARS-CoV-2 replication. Together, these data suggest that repurposing or developing novel host-kinase directed therapies may be an efficacious strategy to prevent or treat COVID-19 and other coronavirus-mediated diseases.

7.
Viruses ; 10(3)2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29518929

RESUMO

Infection of primary CD4+ T cells with HIV-1 coincides with an increase in glycolysis. We investigated the expression of glucose transporters (GLUT) and glycolytic enzymes in human CD4+ T cells in response to infection with HIV-1. We demonstrate the co-expression of GLUT1, GLUT3, GLUT4, and GLUT6 in human CD4+ T cells after activation, and their concerted overexpression in HIV-1 infected cells. The investigation of glycolytic enzymes demonstrated activation-dependent expression of hexokinases HK1 and HK2 in human CD4+ T cells, and a highly significant increase in cellular hexokinase enzyme activity in response to infection with HIV-1. HIV-1 infected CD4+ T cells showed a marked increase in expression of HK1, as well as the functionally related voltage-dependent anion channel (VDAC) protein, but not HK2. The elevation of GLUT, HK1, and VDAC expression in HIV-1 infected cells mirrored replication kinetics and was dependent on virus replication, as evidenced by the use of reverse transcription inhibitors. Finally, we demonstrated that the upregulation of HK1 in HIV-1 infected CD4+ T cells is independent of the viral accessory proteins Vpu, Vif, Nef, and Vpr. Though these data are consistent with HIV-1 dependency on CD4+ T cell glucose metabolism, a cellular response mechanism to infection cannot be ruled out.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Glucose/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/imunologia , Hexoquinase/metabolismo , Biomarcadores , Linfócitos T CD4-Positivos/virologia , Ativação Enzimática , Expressão Gênica , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Glicólise , Infecções por HIV/virologia , Humanos , Ativação Linfocitária/imunologia , Regulação para Cima
8.
AIDS Res Hum Retroviruses ; 33(12): 1236-1247, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28844150

RESUMO

Glutamine is a conditionally essential amino acid that is an important metabolic resource for proliferating tissues by acting as a proteinogenic amino acid, a nitrogen donor for biosynthetic reactions and as a substrate for the citric acid or tricarboxylic acid cycle. The human immunodeficiency virus type 1 (HIV-1) productively infects activated CD4+ T cells that are known to require glutamine for proliferation and for carrying out effector functions. As a virus, HIV-1 is furthermore entirely dependent on host metabolism to support its replication. In this study, we compared HIV-1 infected with uninfected activated primary human CD4+ T cells with regard to glutamine metabolism. We report that glutamine concentrations are elevated in HIV-1-infected cells and that glutamine is important to support HIV-1 replication, although the latter is closely linked to the glutamine dependency of cell survival. Metabolic tracer experiments showed that entry of glutamine-derived carbon into the citric acid cycle is unaffected by HIV-1 infection, but that there is an increase in the secretion of glutamine-derived glutamic acid from HIV-1-infected cells. Western blotting of key enzymes that metabolize glutamine revealed marked differences in the expression of glutaminase isoforms, KGA and CAG, as well as the PPAT enzyme that targets glutamine-derived nitrogen toward nucleotide synthesis. Altogether, this demonstrates that infection of CD4+ T cells with HIV-1 leads to considerable changes in the cellular glutamine metabolism.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Glutamina/metabolismo , Infecções por HIV/patologia , HIV-1/crescimento & desenvolvimento , Replicação Viral/fisiologia , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Infecções por HIV/virologia , Voluntários Saudáveis , Humanos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA