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1.
Anim Sci J ; 91(1): e13378, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32329195

RESUMO

Interferon-tau (IFNT) regulates maternal recognition during early pregnancy in ruminants. The liver can serve as a hematopoietic organ, and it has immune functions. This study hypothesized whether mRNA and proteins of interferon-stimulated genes (ISGs) induced by early pregnancy are upregulated in maternal liver. Therefore, we determined the expression of interferon-stimulated gene 15-kDa protein (ISG15), 2',5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance protein 1 (MX1), interferon-gamma-inducible protein 10 (IP-10), and signal transducer and activator of transcription 1 (STAT1) in maternal livers during early pregnancy in sheep. Ovine livers were sampled on day 16 of the estrous cycle, and days 13, 16, and 25 of pregnancy, and expression of ISGs was detected by quantitative real-time PCR, Western blot, and immunohistochemistry analysis. Our results showed that there were increases in expression of the mRNA and proteins of ISG15, OAS1, IP-10, STAT1, and MX1 during early pregnancy. STAT1 protein was limited to the hepatocytes, and endothelial cells of proper hepatic arteries and hepatic portal veins. In conclusion, the upregulation of ISG15, OAS1, IP-10, STAT1, and MX1 proteins may be implicated in maternal hepatic immune adjustment and other functions during early pregnancy in sheep.


Assuntos
2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Fígado/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Prenhez/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Ovinos/genética , Ovinos/fisiologia , Ubiquitinas/genética , Ubiquitinas/metabolismo , Animais , Ciclo Estral/genética , Ciclo Estral/metabolismo , Feminino , Fígado/imunologia , Gravidez , Prenhez/imunologia , Ovinos/imunologia , Regulação para Cima
2.
Nat Commun ; 11(1): 1048, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32102993

RESUMO

The membrane deforming dynamin family members MxA and MxB are large GTPases that convey resistance to a variety of infectious viruses. During viral infection, Mx proteins are known to show markedly increased expression via an interferon-responsive promoter to associate with nuclear pores. In this study we report that MxB is an inner mitochondrial membrane GTPase that plays an important role in the morphology and function of this organelle. Expression of mutant MxB or siRNA knockdown of MxB leads to fragmented mitochondria with disrupted inner membranes that are unable to maintain a proton gradient, while expelling their nucleoid-based genome into the cytoplasm. These findings implicate a dynamin family member in mitochondrial-based changes frequently observed during an interferon-based, anti-viral response.


Assuntos
DNA Mitocondrial/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Dinaminas/genética , Dinaminas/metabolismo , Células HeLa , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas , Proteínas de Resistência a Myxovirus/genética , Interferência de RNA , RNA Interferente Pequeno/genética
3.
Cell Stem Cell ; 25(6): 784-796.e5, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31809737

RESUMO

The periosteum is critical for bone maintenance and healing. However, the in vivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible for lifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration in vivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.


Assuntos
Actinas/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Periósteo/citologia , Periósteo/metabolismo , Células-Tronco/metabolismo , Actinas/genética , Adolescente , Adulto , Animais , Movimento Celular/fisiologia , Criança , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Masculino , Camundongos Endogâmicos C57BL , Análise em Microsséries , Proteínas de Resistência a Myxovirus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia , Adulto Jovem
4.
Viruses ; 11(12)2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810278

RESUMO

Interferon-mediated host factors myxovirus (Mx) proteins are key features in regulating influenza A virus (IAV) infections. Viral polymerases are essential for viral replication. The Mx1 protein has been known to interact with viral nucleoprotein (NP) and PB2, resulting in the influence of polymerase activity and providing interspecies restriction. The equine influenza virus has evolved as an independent lineage to influenza viruses from other species. We estimated the differences in antiviral activities between human MxA (huMxA) and equine Mx1 (eqMx1) against a broad range of IAV strains. We found that huMxA has antiviral potential against IAV strains from non-human species, whereas eqMx1 could only inhibit the polymerase activity of non-equine species. Here, we demonstrated that NP is the main target of eqMx1. Subsequently, we found adaptive mutations in the NP of strains A/equine/Jilin/1/1989 (H3N8JL89) and A/chicken/Zhejiang/DTID-ZJU01/2013 (H7N9ZJ13) that confer eqMx1 resistance and sensitivity respectively. A substantial reduction in Mx1 resistance was observed for the two mutations G34S and H52N in H3N8JL89 NP. Thus, eqMx1 is an important dynamic force in IAV nucleoprotein evolution. We, therefore, suggest that the amino acids responsible for Mx1 resistance should be regarded as a robust indicator for the pandemic potential of lately evolving IAVs.


Assuntos
Doenças dos Cavalos/metabolismo , Doenças dos Cavalos/virologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A/fisiologia , Proteínas de Resistência a Myxovirus/metabolismo , Infecções por Orthomyxoviridae/veterinária , Replicação Viral , Animais , Cães , Evolução Molecular , Células HEK293 , Cavalos , Humanos , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Mutação , Proteínas do Nucleocapsídeo , Filogenia , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Especificidade da Espécie , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
5.
Cell Rep ; 29(7): 1923-1933.e3, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31722207

RESUMO

Myxovirus resistance 2 (MX2/MXB) is an interferon (IFN)-induced HIV-1 restriction factor that inhibits viral nuclear DNA accumulation. The amino-terminal domain of MX2 binds the viral capsid and is essential for inhibition. Using in vitro assembled Capsid-Nucleocapsid (CANC) complexes as a surrogate for the HIV-1 capsid lattice, we reveal that the GTPase (G) domain of MX2 contains a second, independent capsid-binding site. The importance of this interaction was addressed in competition assays using the naturally occurring non-antiviral short isoform of MX2 that lacks the amino-terminal 25 amino acids. Specifically, these experiments show that the G domain enhances MX2 function, and the foreshortened isoform acts as a functional suppressor of the full-length protein in a G-domain-dependent manner. The interaction of MX2 with its HIV-1 capsid substrate is therefore multi-faceted: there are dual points of contact that, together with protein oligomerization, contribute to the complexity of MX2 regulation.


Assuntos
Capsídeo/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Infecções por HIV/genética , HIV-1/genética , Células HeLa , Humanos , Proteínas de Resistência a Myxovirus/genética , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
6.
PLoS Biol ; 17(10): e3000181, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31574080

RESUMO

Antagonistic interactions drive host-virus evolutionary arms races, which often manifest as recurrent amino acid changes (i.e., positive selection) at their protein-protein interaction interfaces. Here, we investigated whether combinatorial mutagenesis of positions under positive selection in a host antiviral protein could enhance its restrictive properties. We tested approximately 700 variants of human MxA, generated by combinatorial mutagenesis, for their ability to restrict Thogotovirus (THOV). We identified MxA super-restrictors with increased binding to the THOV nucleoprotein (NP) target protein and 10-fold higher anti-THOV restriction relative to wild-type human MxA, the most potent naturally occurring anti-THOV restrictor identified. Our findings reveal a means to elicit super-restrictor antiviral proteins by leveraging signatures of positive selection. Although some MxA super-restrictors of THOV were impaired in their restriction of H5N1 influenza A virus (IAV), other super-restrictor variants increased THOV restriction without impairment of IAV restriction. Thus, broadly acting antiviral proteins such as MxA mitigate breadth-versus-specificity trade-offs that could otherwise constrain their adaptive landscape.


Assuntos
Virus da Influenza A Subtipo H5N1/genética , Proteínas de Resistência a Myxovirus/genética , Nucleoproteínas/genética , Thogotovirus/genética , Proteínas Virais/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Evolução Molecular , Regulação da Expressão Gênica , Biblioteca Gênica , Células HEK293 , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Especificidade de Hospedeiro , Humanos , Virus da Influenza A Subtipo H5N1/metabolismo , Mutagênese , Proteínas de Resistência a Myxovirus/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Nucleoproteínas/metabolismo , Transdução de Sinais , Thogotovirus/metabolismo , Proteínas Virais/metabolismo
7.
Microb Pathog ; 137: 103749, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31521801

RESUMO

Human respiratory syncytial virus (RSV) is the most common viral pathogen of lower respiratory tract infection worldwide. The virus selectively infects the respiratory epithelium, and causes diseases of variable severity in infants and the elderly. However, the differences in pathogenesis in the age groups remain poorly studied. Age is a major determinant of RSV disease, and the most severe morbidity and mortality occur in the infants and the elderly, because of the immature immunity in infants and declining immunity in old age. The cotton rat is a good model of RSV infection as it is naturally susceptible to RSV. In this study, we established an infant/adult/elderly RSV infection model in 3-week, 8-week and 30-week-old cotton rats and infected them with equal dose of RSV. This model exhibited airway neutrophils infiltration. In the 3-week-old group, higher viral load was observed in the lungs and noses, may due to low IFN-α/Mx2 levels. In contrast, the 8-week-old group had adequate IFN-α/Mx2 but exhibited the most obvious pulmonary inflammation and peribronchiolitis. Interestingly, the most severe pathology and delayed viral clearance in the lungs were observed in the 30-week-old group, may related to the increase of mucus induced by TNF-α and the lower antiviral effect of IFN-α. These results clearly revealed that an age-dependent severity of RSV disease and antiviral defense in the cotton rats, which may provide an effective model for personalized vaccine research and specific treatment strategies for different RSV age groups.


Assuntos
Pulmão/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/patogenicidade , Animais , Antivirais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Feminino , Imunidade Inata , Interferon-alfa/metabolismo , Pulmão/patologia , Pulmão/virologia , Proteínas de Resistência a Myxovirus/metabolismo , Sigmodontinae , Carga Viral
8.
J Virol ; 93(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31484749

RESUMO

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g., stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membraneless metastable (shape-changing) condensates in the cytoplasm. In our studies, we used the same cell lines and methods as those used by previous investigators but concluded that wild-type MxA formed variably sized spherical or irregular bodies, filaments, and even a reticulum distinct from that of ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of green fluorescent protein (GFP)-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24 ± 0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase, respectively, in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature and provide evidence for membraneless MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation and further report that MxA forms membraneless metastable (shape-changing) condensates in the cytoplasm consisting of variably sized spherical or irregular bodies, filaments, and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1 to 3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.


Assuntos
Citoplasma/virologia , Proteínas de Resistência a Myxovirus/metabolismo , Linhagem Celular , Efeito Citopatogênico Viral/fisiologia , Citoplasma/metabolismo , Humanos , Orthomyxoviridae/metabolismo , Proteínas/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Viroses/metabolismo , Vírus/metabolismo
9.
Nat Commun ; 10(1): 3753, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434895

RESUMO

Semen is the vehicle for virion dissemination in the female reproductive tract (FRT) in male-to-female HIV transmission. Recent data suggests that higher frequency semen exposure is associated with activation of anti-HIV mechanisms in HIV negative sex workers. Here, we use a non-human primate (NHP) model to show that repeated vaginal exposure to semen significantly reduces subsequent infection by repeated low-dose vaginal SIVmac251 challenge. Repeated semen exposures result in lower CCR5 expression in circulating CD4+ T-cells, as well as higher expression of Mx1 (in correlation with IFNε expression) and FoxP3 in the cervicovaginal mucosa, and increased infiltration of CD4+ T-cells. Establishing in vivo evidence of competing effects of semen on transmission impacts our basic understanding of what factors may determine HIV infectivity in humans. Our results clearly indicate that repeated semen exposure can profoundly modulate the FRT microenvironment, paradoxically promoting host resistance against HIV acquisition.


Assuntos
Colo do Útero/imunologia , Membrana Mucosa/imunologia , Sêmen/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/imunologia , Vagina/imunologia , Animais , Linfócitos T CD4-Positivos , Colo do Útero/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/transmissão , Humanos , Macaca mulatta , Membrana Mucosa/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Receptores CCR5/metabolismo , Vagina/virologia
10.
Mult Scler Relat Disord ; 35: 241-245, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421628

RESUMO

BACKGROUND: Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system (CNS) characterized by immune-mediated demyelination and axonal injury. Myelin-reactive IFN-γ-producing Th1 cells has been shown to play an important role in the development of MS. MicroRNAs (miRNAs) are a new class of small non-coding RNA molecules about 22 nucleotides long which regulate gene expression post-transcriptionally by binding to 3' UTR of their mRNA targets, and resulting in degradation or transcriptional repression of the targeted mRNA. Accumulating evidence supports that miRNA dysregulation is linked to the pathogenesis of autoimmune diseases that include MS. miR-29b expression has been shown to be upregulated in memory CD4+T cells from relapsing-remitting MS (RR-MS) patients, which may reflect chronic Th1 inflammation. Interferon beta (IFN-ß) benefits patients with MS and reduces symptoms of the RR-MS. MxA is induced by type I interferon and predicts IFN-ß response in MS patients. The aim of this study was to evaluate miR-29b variants and MxA expression and serum IFN-γ level in responders and non-responders to IFN-ß treatment. METHODS: A total of 70 IFN-ß treated RR-MS patients including 35 responders and 35 non-responders were enrolled. We analyzed the expression level of miR-29b variants and MxA using the peripheral blood of MS patients treated with IFN-ß for more than one year. Real-time RT-PCR was performed to analyze miR-29b variants and MxA expression one year after initiation of IFN-ß therapy. Serum cytokine level was measured by ELISA. RESULTS: The results indicated that the expression level of miR-29b-3p changed related to IFN-ß response. Moreover, miR-29b-5p was downregulated under IFN-ß treatment in responders versus non-responders. MxA level was significantly decreased in the responders. There was no change in IFN-γ level following treatment with IFN-ß in the MS patients. CONCLUSIONS: Our results might provide fundamentals for the development of new markers of the biological effects of IFN-ß therapy.


Assuntos
Fatores Imunológicos/uso terapêutico , Interferon beta/uso terapêutico , MicroRNAs/metabolismo , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Proteínas de Resistência a Myxovirus/metabolismo , Adulto , Citocinas/sangue , Feminino , Humanos , Masculino , MicroRNAs/genética , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla Recidivante-Remitente/metabolismo , Proteínas de Resistência a Myxovirus/genética , Resultado do Tratamento , Adulto Jovem
11.
J Cell Biochem ; 120(11): 18762-18770, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31265172

RESUMO

In human, there are two myxovirus resistance genes-MX1 and MX2, which respectively encode MXA and MXB protein. For MXB, it was traditionally deemed to work in the progression of cell cycle and adjustment of nuclear import. Thus, we speculated that it might play important roles in tumor progression. The purpose of this study was to preliminarily explore the underlying functions and mechanism of the MX2 gene on glioblastoma multiforme. Quantitative reverse transcription polymerase chain reaction, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), and transwell experiments were to detect the relative MX2 mRNA level and its biological functions on glioma cells, respectively. The data displayed that MX2 was obviously downregulated both in glioblastoma (GBM) and GBM cell lines, meanwhile, its overexpression could markedly reduce cell proliferation, migration, and invasion of glioma cells, implying that it was related with glioblastoma progression. In addition, the overall survival of patient with glioblastoma had a negative correlation with the MX2 expression. Then, Western blot indicated the potential mechanism of MX2 in glioblastoma. We found that MX2 overexpression could decrease the relative levels of phosphorylated-ERK1/2 (p-ERK1/2), p-p38, and nuclear factor-κB (NF-κB), while have no effects on extracellular signal-regulated kinase (ERK), p38, and lamin B1. Moreover, the influences of MX2 overexpression on cell proliferation, migration, and invasion could be weakened by the three inhibitors (PD98059, SB203580, and (pyridin-2-ylmethyl) dithiocarbamate [PDTC]). These results implied that MX2 might suppress the proliferation and metastasis of glioma cells by manipulating the ERK/P38/NF-κB signaling pathway. In conclusion, MX2 is potential to be a new marker used for glioblastoma prognosis or a new target for glioblastoma treatments.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioblastoma/genética , Proteínas de Resistência a Myxovirus/genética , NF-kappa B/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Imidazóis/farmacologia , Proteínas de Resistência a Myxovirus/metabolismo , Invasividade Neoplásica , Fosforilação , Prolina/análogos & derivados , Prolina/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiocarbamatos/farmacologia
12.
Int J Biol Macromol ; 136: 1258-1272, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31242450

RESUMO

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well.


Assuntos
Cipriniformes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Guanosina Trifosfato/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Proteínas de Peixes/química , Cinética , Simulação de Dinâmica Molecular , Proteínas de Resistência a Myxovirus/química , Filogenia , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , RNA Mensageiro/genética , Termodinâmica
13.
Structure ; 27(8): 1234-1245.e5, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31155311

RESUMO

The human antiviral protein MxB is a restriction factor that fights HIV infection. Previous experiments have demonstrated that MxB targets the HIV capsid, a protein shell that protects the viral genome. To make the conical-shaped capsid, HIV CA proteins are organized into a lattice composed of hexamer and pentamer building blocks, providing many interfaces for host proteins to recognize. Through extensive biochemical and biophysical studies and molecular dynamics simulations, we show that MxB is targeting the HIV capsid by recognizing the region created at the intersection of three CA hexamers. We are further able to map this interaction to a few CA residues, located in a negatively charged well at the interface between the three CA hexamers. This work provides detailed residue-level mapping of the targeted capsid interface and how MxB interacts. This information could inspire the development of capsid-targeting therapies for HIV.


Assuntos
Capsídeo/química , Capsídeo/metabolismo , HIV-1/metabolismo , Proteínas de Resistência a Myxovirus/química , Proteínas de Resistência a Myxovirus/metabolismo , Sítios de Ligação , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Proteínas de Resistência a Myxovirus/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica
14.
J Virol ; 93(15)2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31092574

RESUMO

Myxovirus resistance 1 (Mx1) is an interferon-induced gene that encodes a GTPase that plays an important role in the defense of mammalian cells against influenza A and other viruses. The Mx1 protein can restrict a number of viruses independently of the expression of other interferon-induced genes. Mx genes are therefore considered to be an important part of the innate antiviral immune response. However, the possible impact of Mx expression in the hematopoietic cellular compartment has not been investigated in detail in the course of a viral infection. To address this, we performed bone marrow chimera experiments using congenic B6.A2G Mx1 +/+ and B6.A2G Mx1-/- mice to study the effect of Mx1 expression in cells of hematopoietic versus nonhematopoietic origin. Mx1+/+ mice were protected and Mx1-/- mice were susceptible to influenza A virus challenge infection, regardless of the type of bone marrow cells (Mx1 +/+ or Mx1-/- ) the animals had received. Infection with Thogoto virus, however, revealed that Mx1-/- mice with a functional Mx1 gene in the bone marrow compartment showed reduced liver pathology compared with Mx1-/- mice that had been grafted with Mx1 -/- bone marrow. The reduced pathology in these mice was associated with a reduction in Thogoto virus titers in the spleen, lung, and serum. Moreover, Mx1 +/+ mice with Mx1 -/- bone marrow failed to control Thogoto virus replication in the spleen. Mx1 in the hematopoietic cellular compartment thus contributes to protection against Thogoto virus infection.IMPORTANCE Mx proteins are evolutionarily conserved in vertebrates and can restrict a wide range of viruses in a cell-autonomous way. The contribution to antiviral defense of Mx1 expression in hematopoietic cells remains largely unknown. We show that protection against influenza virus infection requires Mx1 expression in the nonhematopoietic cellular compartment. In contrast, Mx1 in bone marrow-derived cells is sufficient to control disease and virus replication following infection with a Thogoto virus. This indicates that, in addition to its well-established antiviral activity in nonhematopoietic cells, Mx1 in hematopoietic cells can also play an important antiviral function. In addition, cells of hematopoietic origin that lack a functional Mx1 gene contribute to Thogoto virus dissemination and associated disease.


Assuntos
Células da Medula Óssea/imunologia , Imunidade Inata , Fatores Imunológicos/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Infecções por Orthomyxoviridae/imunologia , Thogotovirus/imunologia , Animais , Medula Óssea/virologia , Fatores Imunológicos/deficiência , Vírus da Influenza A/imunologia , Pulmão/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Resistência a Myxovirus/deficiência , Infecções por Orthomyxoviridae/patologia , Soro/virologia , Baço/virologia , Carga Viral
15.
J Interferon Cytokine Res ; 39(5): 274-282, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30939061

RESUMO

Type I/III interferons provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced, Mx proteins of some species appear as key components of defense against influenza A viruses. It is expected that such an antiviral protein must display a platform dedicated to the recognition of said viruses. In an attempt to identify such platform in human MxA, an evolution-guided approach capitalizing on the antagonistic arms race between MxA and its viral targets and the genomic signature it left on primate genomes revealed that the surface-exposed so-called "loop L4", which protrudes from the compact structure of the MxA stalk, is a hotspot of recurrent positive selection. Since MxA is archetypic of Mx1 proteins in general, we reasoned that the L4 loop also functions as a recognition platform for influenza viruses in the Mx1 proteins of other species that had been exposed to the virus for ever. In this study, the anti-influenza activity of 5 distinct mammalian Mx1 proteins was measured by comparing the number of viral nucleoprotein-positive cells 7 h after infection in a sample of 100,000 cells expected to contain both Mx1-positive and Mx1-negative cell subpopulations. The systematic depletion (P < 0.001) of virus nucleoprotein-positive cells among equine, bubaline, porcine, and bovine Mx1-expressing cell populations compared with Mx-negative cells suggests a strong anti-influenza A activity. Looking for common anti-influenza signature elements in the sequence of these Mx proteins, we found that an aromatic residue at positions 561 or 562 in the L4 loop seems critical for the anti-influenza function and/or specificity of mammalian Mx1.


Assuntos
Vírus da Influenza A/imunologia , Interferon Tipo I/imunologia , Interferons/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Animais , Búfalos , Bovinos , Cães , Células HEK293 , Cavalos , Humanos , Suínos
16.
Int J Mol Sci ; 20(7)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939763

RESUMO

The demyelinating canine distemper virus (CDV)-leukoencephalitis represents a translational animal model for multiple sclerosis. The present study investigated the expression of type I interferon (IFN-I) pathway members in CDV-induced cerebellar lesions to gain an insight into their role in lesion development. Gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. Interferon regulatory factor (IRF) 3, IRF7, signal transducer and activator of transcription (STAT) 1, STAT2, MX protein, protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (OAS) 1 and interferon-stimulated gene (ISG) 15 expression were also evaluated using immunohistochemistry. Cellular origin of STAT1, STAT2, MX and PKR were determined using immunofluorescence. CDV infection caused an increased expression of the antiviral effector proteins MX, PKR, OAS1 and ISG15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. This increase might be partly mediated by IRF-dependent pathways due to the lack of changes in IFN-I levels and absence of STAT2 in astrocytes. Nevertheless, activated microglia/macrophages showed a strong expression of STAT1, STAT2 and MX proteins in later stages of the disease, indicating a strong activation of the IFN-I signaling cascade, which might be involved in the aggravation of bystander demyelination.


Assuntos
Cinomose/genética , Imunidade Inata/genética , Interferons/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cinomose/imunologia , Cães , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
17.
Virology ; 531: 260-268, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30959264

RESUMO

SAMHD1 is a human restriction factor known to prevent infection of macrophages, resting CD4+ T cells, and dendritic cells by HIV-1. To test the contribution of MxB to the ability of SAMHD1 to block HIV-1 infection, we created human THP-1 cell lines that were knocked out for expression of MxB, SAMHD1, or both. Interestingly, MxB depletion renders SAMHD1 ineffective against HIV-1 but not SIVmac. We observed similar results in human primary macrophages that were knockdown for the expression of MxB. To understand how MxB assists SAMHD1 restriction of HIV-1, we examined direct interaction between SAMHD1 and MxB in pull-down experiments. In addition, we investigated several properties of SAMHD1 in the absence of MxB expression, including subcellular localization, phosphorylation of the SAMHD1 residue T592, and dNTPs levels. These experiments showed that SAMHD1 restriction of HIV-1 requires expression of MxB.


Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Proteínas de Resistência a Myxovirus/metabolismo , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Vírus da Imunodeficiência Símia/fisiologia , Motivos de Aminoácidos , Infecções por HIV/genética , Infecções por HIV/virologia , Humanos , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas de Resistência a Myxovirus/genética , Fosforilação , Ligação Proteica , Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/genética , Especificidade da Espécie
18.
Cell Stem Cell ; 24(6): 944-957.e5, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006621

RESUMO

Stem cell heterogeneity is recognized as functionally relevant for tissue homeostasis and repair. The identity, context dependence, and regulation of skeletal muscle satellite cell (SC) subsets remains poorly understood. We identify a minor subset of Pax7+ SCs that is indelibly marked by an inducible Mx1-Cre transgene in vivo, is enriched for Pax3 expression, and has reduced ROS (reactive oxygen species) levels. Mx1+ SCs possess potent stem cell activity upon transplantation but minimally contribute to endogenous muscle repair, due to their relative low abundance. In contrast, a dramatic clonal expansion of Mx1+ SCs allows extensive contribution to muscle repair and niche repopulation upon selective pressure of radiation stress, consistent with reserve stem cell (RSC) properties. Loss of Pax3 in RSCs increased ROS content and diminished survival and stress tolerance. These observations demonstrate that the Pax7+ SC pool contains a discrete population of radiotolerant RSCs that undergo clonal expansion under severe stress.


Assuntos
Células-Tronco Adultas/fisiologia , Dano ao DNA/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular , Células Clonais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Resistência a Myxovirus/metabolismo , Fator de Transcrição PAX3/metabolismo , Fator de Transcrição PAX7/metabolismo , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismo , Regeneração , Regulação para Cima
19.
PLoS One ; 14(3): e0214319, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30913245

RESUMO

Porcine gamma interferon is a cytokine produced by activated T cells and NK cells with broad-spectrum antiviral activity and immunomodulatory function. However, pIFN-γ is a secretory protein that has a short half-life in organisms and is easily inactivated, making it difficult to apply widely in clinics. Therefore, we tried to optimize the expression of pIFN-γ in Pichia pastoris to obtain a large amount of highly active, easily purified pIFN-γ protein in vitro. Through C-terminal sequence analysis, we found a signal sequence (EKREAEAE) that was easily enzymolysed by a signal peptide enzyme, resulting in degradation and inactivation of the pIFN-γ protein. In this study, we optimized the pIFN-γ gene recombination sequence and mutated the 3' end of the pIFN-γ gene, resulting in a higher expression level and stronger biological activity, as well as a significant upregulation in the expression of the interferon-stimulated genes Mx1 and OAS1 in IPEC-J2 jejunal epithelial cells. Our data also showed that the fermentation process could significantly improve productivity. A recombinant Pichia pastoris strain with the optimized pIFN-γ gene could obtain a high yield of pIFN-γ protein, up to 9536 mg/L, after staged incubation for 0-24 h at 28°C, pH 6.0, and 50% dissolved oxygen (DO), followed by incubation for 24-72 h at 25°C, pH 6.0 and 30% DO. These data demonstrated, for the first time, that the expression level of pIFN-γ in Pichia pastoris was improved significantly by gene optimization with 3' end mutation and a fermentation process that maintained good biological activity, which is beneficial to the application of pIFN-γ in animal husbandry.


Assuntos
Interferon gama/metabolismo , Pichia/metabolismo , Sinais Direcionadores de Proteínas/genética , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Técnicas de Cultura Celular por Lotes , Linhagem Celular , Histidina/genética , Histidina/metabolismo , Interferon gama/genética , Mutação , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Suínos
20.
Sci Rep ; 9(1): 3956, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850653

RESUMO

The myxovirus resistance (Mx) proteins belong to interferon-induced dynamin GTPase and play pivotal role in the inhibition of replication of numerous viruses. These antiviral proteins are released in usual or diseased condition to prevent the viral attack and to carry regular cellular activities like endocytosis and trafficking of nucleoproteins into the nucleus. The invasion of virus up-regulates the expression of Mx transcripts and double-stranded RNA mimic like polyinosinic polycytidyilic acid (Poly I:C). To understand the tissue-specific expression profiling and mechanism of GTP recognition of Mx protein from Labeo rohita (rohu), the full-length gene was cloned, sequenced and characterized through various Bioinformatics tools for the first time. The Mx cDNA was comprised of 2297 bp, and the open reading frame of 1938 bp encodes polypeptide of 631 amino acids. The coding sequence of Mx protein possess the signature motif of dynamin superfamily, LPRG(S/K)GIVTR, the tripartite guanosine-5/triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD) and the leucine zipper motifs at the C-terminal end, well conserved in all interferon-induced Mx protein in vertebrates. Western blotting confirmed the molecular weight of Mx protein to be 72 kDa. After the intraperitoneal challenge of L. rohita with a Poly I:C, up-regulation of Mx protein was observed in brain, spleen, liver, kidney, intestine, heart, muscle, and gill. Ontogeny study displayed pronounced expression of Mx protein in all stages of the developmental of Rohu after Poly I:C induction. However a persistent expression of Mx transcript was also observed in Rohu egg as well as milt without induction with Poly I:C. Higher expression of Mx gene was observed on 96 h where it was 6.4 folds higher than the control. The computational modelling of Mx protein portrayed the tripartite N-terminal G-domain that binds to GTP, the bundle-signaling element (BSE) which interconnects the G-domain to the elongated stalk domain and C-terminal helical stalk domain. In agreement with the experimental studies, a series of conserved residues viz., Gln52, Ser53, Ser54, Leu68, Pro69, Gly71, Gly73, Thr76, Asp151, Gly154, Thr220, Lys221, Val251, Cys253, Arg254, and Gly255 were computed to be indispensable for tight anchoring of GTP within binding cavity of G-domain. The binding free energy calculation study depicted that the van der Waals and electrostatic terms contributs significantly to molecular recognition of GTP. Collectively, our study provides mechanistic insights into the tissue-specific expression profiling and GTP binding mechanism of Mx protein from Labeo rohita, which is expected to drive further research on several cellular events including viral resistance and endocytosis in the near future.


Assuntos
Cyprinidae/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , Poli I-C/farmacologia , Animais , Clonagem Molecular/métodos , Cyprinidae/virologia , Feminino , GTP Fosfo-Hidrolases/metabolismo , Masculino , Proteínas de Resistência a Myxovirus/genética , Orthomyxoviridae , Óvulo/metabolismo , Filogenia , Domínios Proteicos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Distribuição Tecidual , Transcriptoma/efeitos dos fármacos
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