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1.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638901

RESUMO

Among the mechanisms leading to progression to Adult T-cell Leukaemia/Lymphoma in Human T-cell Leukaemia Virus type 1 (HTLV-1)-infected subjects, the contribution of stromal components remains poorly understood. To dissect the role of fibroblasts in HTLV-1-mediated lymphomagenesis, transcriptome studies, cytofluorimetric and qRT-PCR analyses of surface and intracellular markers linked to plasticity and stemness in coculture, and in vivo experiments were performed. A transcriptomic comparison between a more lymphomagenic (C91/III) and the parental (C91/PL) cell line evidenced hyperactivation of the PI3K/Akt pathway, confirmed by phospho-ELISA and 2-DE and WB analyses. C91/III cells also showed higher expression of mesenchymal and stemness genes. Short-term coculture with human foreskin fibroblasts (HFF) induced these features in C91/PL cells, and significantly increased not only the cancer stem cells (CSCs)-supporting CD10+GPR77+ HFF subpopulation, but also the percentage of ALDH1bright C91/PL cells. A non-cytotoxic acetylsalicylic acid treatment decreased HFF-induced ALDH1bright C91/PL cells, downregulated mesenchymal and stemness genes in cocultured cells, and delayed lymphoma growth in immunosuppressed mice, thus hindering the supportive activity of HFF on CSCs. These data suggest that crosstalk with HFF significantly intensifies the aggressiveness and plasticity of C91/PL cells, leading to the enrichment in lymphoma-initiating cells. Additional research is needed to better characterize these preliminary findings.


Assuntos
Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Linfoma/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/farmacologia , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , Linfoma/tratamento farmacológico , Linfoma/virologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/virologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
2.
J Gen Virol ; 102(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34546869

RESUMO

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/metabolismo , Encefalite Japonesa/virologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Proteoma , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Colágeno/genética , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo , Encefalite Japonesa/genética , Encefalite Japonesa/imunologia , Fibroblastos/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Inflamação , Interferons/imunologia , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Proteínas/metabolismo , Proteômica , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Regulação para Cima
3.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575976

RESUMO

Human cytomegalovirus (HCMV) infects 40-70% of adults in developed countries. HCMV proteins and DNA are detected in tumors and metastases, suggesting an association with increased invasion. We investigated HCMV infection in human breast cancer cell lines compared to fibroblasts, a component of tumors, and the role of platelet-derived growth factor receptor-α (PDGFRα). HCMV productively infected HEL299 fibroblasts and, to a lesser extent, Hs578T breast cancer cells. Infection of another triple-negative cell line, MDA-MB-231, and also MCF-7 cells, was extremely low. These disparate infection rates correlated with expression of PDGFRA, which facilitates HCMV uptake. Increasing PDGFRA expression in T-47D breast cancer and BCPAP thyroid cancer cells markedly increased HCMV infection. Conversely, HCMV infection decreased PDGFRA expression, potentially attenuating signaling through this receptor. HCMV infection of fibroblasts promoted the secretion of proinflammatory factors, whereas an overall decreased secretion of inflammatory factors was observed in infected Hs578T cells. We conclude that HCMV infection in tumors will preferentially target tumor-associated fibroblasts and breast cancer cells expressing PDGFRα. HCMV infection in the tumor microenvironment, rather than cancer cells, will increase the inflammatory milieu that could enhance metastasis involving lysophosphatidate.


Assuntos
Neoplasias da Mama/genética , Infecções por Citomegalovirus/genética , Lisofosfolipídeos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Neoplasias da Mama/virologia , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/complicações , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Feminino , Fibroblastos/patologia , Fibroblastos/virologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Lisofosfolipídeos/metabolismo , Células MCF-7 , Metástase Neoplásica/genética , Transdução de Sinais/genética , Microambiente Tumoral/genética , Internalização do Vírus
4.
PLoS Pathog ; 17(8): e1009803, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34352038

RESUMO

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.


Assuntos
Membrana Celular/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Glicosaminoglicanos/química , Heparitina Sulfato/química , Polimerização , Internalização do Vírus , Animais , Membrana Celular/virologia , Infecções por Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Vírion
5.
Cells ; 10(7)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34359995

RESUMO

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.


Assuntos
Vírus Chikungunya/genética , Receptor Celular 1 do Vírus da Hepatite A/genética , Interações Hospedeiro-Patógeno/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores Virais/genética , Internalização do Vírus , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/crescimento & desenvolvimento , Vírus Chikungunya/imunologia , Chlorocebus aethiops , Cricetulus , Endossomos/efeitos dos fármacos , Endossomos/imunologia , Endossomos/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Expressão Gênica , Células HEK293 , Receptor Celular 1 do Vírus da Hepatite A/antagonistas & inibidores , Receptor Celular 1 do Vírus da Hepatite A/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Queratinócitos/imunologia , Queratinócitos/virologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/imunologia , Receptores Virais/antagonistas & inibidores , Receptores Virais/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transgenes , Células Vero , Internalização do Vírus/efeitos dos fármacos
6.
Bioengineered ; 12(1): 4407-4419, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34436976

RESUMO

Widespread infection due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has led to a global pandemic. Currently, various approaches are being taken up to develop vaccines and therapeutics to treat SARS-CoV2 infection. Consequently, the S protein has become an important target protein for developing vaccines and therapeutics against SARS-CoV2. However, the highly infective nature of SARS-CoV2 restricts experimentation with the virus to highly secure BSL3 facilities. The availability of fusion-enabled, nonreplicating, and nonbiohazardous mimics of SARS-CoV2 virus fusion, containing the viral S or S and M protein in their native conformation on mammalian cells, can serve as a useful substitute for studying viral fusion for testing various inhibitors of viral fusion. This would avoid the use of the BSL3 facility for fusion studies required to develop therapeutics. In the present study, we have developed SARS-CoV2 virus fusion mimics (SCFMs) using mammalian cells transfected with constructs coding for S or S and M protein. The fusogenic property of the mimic(s) and their interaction with the functional human ACE2 receptors was confirmed experimentally. We have also shown that such mimics can easily be used in an inhibition assay. These mimic(s) can be easily prepared on a large scale, and such SCFMs can serve as an invaluable resource for viral fusion inhibition assays and in vitro screening of antiviral agents, which can be shared/handled between labs/facilities without worrying about any biohazard while working under routine laboratory conditions, avoiding the use of BSL3 laboratory.Abbreviations :SCFM: SARS-CoV2 Virus Fusion Mimic; ACE2: Angiotensin-Converting Enzyme 2; hACE2: Human Angiotensin-Converting enzyme 2; MEF: Mouse Embryonic Fibroblasts; HBSS: Hanks Balanced Salt Solution; FBS: Fetal Bovine Serum.


Assuntos
Anticorpos Neutralizantes/farmacologia , Contenção de Riscos Biológicos/métodos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas da Matriz Viral/antagonistas & inibidores , Internalização do Vírus/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Chlorocebus aethiops , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Camundongos , Mimetismo Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Ligação Proteica , Receptores Virais/genética , Receptores Virais/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Transfecção , Células Vero , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
7.
mBio ; 12(4): e0157221, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34372702

RESUMO

Tissue- and cell-specific expression patterns are highly variable within and across individuals, leading to altered host responses after acute virus infection. Unraveling key tissue-specific response patterns provides novel opportunities for defining fundamental mechanisms of virus-host interaction in disease and the identification of critical tissue-specific networks for disease intervention in the lung. Currently, there are no approved therapeutics for Middle East respiratory syndrome coronavirus (MERS-CoV) patients, and little is understood about how lung cell types contribute to disease outcomes. MERS-CoV replicates equivalently in primary human lung microvascular endothelial cells (MVE) and fibroblasts (FB) and to equivalent peak titers but with slower replication kinetics in human airway epithelial cell cultures (HAE). However, only infected MVE demonstrate observable virus-induced cytopathic effect. To explore mechanisms leading to reduced MVE viability, donor-matched human lung MVE, HAE, and FB were infected, and their transcriptomes, proteomes, and lipidomes were monitored over time. Validated functional enrichment analysis demonstrated that MERS-CoV-infected MVE were dying via an unfolded protein response (UPR)-mediated apoptosis. Pharmacologic manipulation of the UPR in MERS-CoV-infected primary lung cells reduced viral titers and in male mice improved respiratory function with accompanying reductions in weight loss, pathological signatures of acute lung injury, and times to recovery. Systems biology analysis and validation studies of global kinetic transcript, protein, and lipid data sets confirmed that inhibition of host stress pathways that are differentially regulated following MERS-CoV infection of different tissue types can alleviate symptom progression to end-stage lung disease commonly seen following emerging coronavirus outbreaks. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe atypical pneumonia in infected individuals, but the underlying mechanisms of pathogenesis remain unknown. While much has been learned from the few reported autopsy cases, an in-depth understanding of the cells targeted by MERS-CoV in the human lung and their relative contribution to disease outcomes is needed. The host response in MERS-CoV-infected primary human lung microvascular endothelial (MVE) cells and fibroblasts (FB) was evaluated over time by analyzing total RNA, proteins, and lipids to determine the cellular pathways modulated postinfection. Findings revealed that MERS-CoV-infected MVE cells die via apoptotic mechanisms downstream of the unfolded protein response (UPR). Interruption of enzymatic processes within the UPR in MERS-CoV-infected male mice reduced disease symptoms, virus-induced lung injury, and time to recovery. These data suggest that the UPR plays an important role in MERS-CoV infection and may represent a host target for therapeutic intervention.


Assuntos
Lesão Pulmonar Aguda/patologia , Apoptose/fisiologia , Infecções por Coronavirus/patologia , Resposta a Proteínas não Dobradas/fisiologia , Lesão Pulmonar Aguda/virologia , Animais , Linhagem Celular , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/virologia , Humanos , Masculino , Camundongos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia
8.
Pol J Vet Sci ; 24(2): 287-292, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34250783

RESUMO

Viral diseases have caused devastating effect on poultry industry leading to significant losses in economy of world. In the presented study, the ability of Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and avian influenza virus (AIV) to grow in two cell lines was evaluated. Both chicken embryo fibroblast (CEF) and DF-1 cells were used and cytopathic effects (CPE) produced by these viruses were observed. The titer of virus in terms of TCID was determined after 24h up to four days for each virus. The same type of CPE was observed for all virus- es used in the study in both DF-1 and CEF cells. IBDV showed CPE causing rounding of cells while NDV caused formation of multicellular large nuclei, cell fusion and rounding of cells. Giant cells with inclusions and aggregation of cells with intact monolayer was observed for AIV. In growth kinetic study, higher titer of IBDV and NDV was observed in CEF cells than DF-1 cells while for AIV, DF-1 cells showed higher titer than CEF cells. These results would be useful for furthers comparative studies on growth of different cell lines of various viruses to find a suitability for vaccine production.


Assuntos
Fibroblastos/virologia , Vírus da Doença Infecciosa da Bursa/fisiologia , Vírus da Influenza A/fisiologia , Vírus da Doença de Newcastle/fisiologia , Replicação Viral/fisiologia , Animais , Linhagem Celular , Embrião de Galinha , Fatores de Tempo , Cultura de Vírus
9.
Vet Microbiol ; 260: 109093, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34265512

RESUMO

The interactions between host cellular proteins and viral proteins are important for successful infection by viruses. Previous studies from our group have identified various host cellular proteins that can interact with the Newcastle disease virus V protein (Chu et al., 2018a), but their function in NDV replication has not been fully determined. The present study reports that heterogenous nuclear ribonucleoprotein H1 (hnRNP H1) can interact with NDV V protein in yeast. The immunofluorescence results showed that hnRNP H1 and V protein could colocalize in the cytoplasm of a chicken embryo fibroblast cell line (DF-1 cells). Co-immunoprecipitation assays further verified the interaction of these two proteins. The effects of overexpression and knockdown of hnRNP H1 on NDV replication were evaluated in DF-1 cells through real time quantitative PCR (RT-qPCR) and plaque assays. The regulation of V protein on hnRNP H1 expression was also examined. The results indicated that overexpression of hnRNP H1 facilitated NDV replication, while knockdown of hnRNP H1 decreased NDV replication. It was also shown that V protein could regulate hnRNP H1 expression at the protein level instead of the transcription level. The effect of V protein and hnRNP H1 on the DF-1 cell cycle was also tested and the results revealed that V protein may regulate cell proliferation by controlling the expression of hnRNP H1. Taken together, these results suggest that NDV V protein could promote viral replication by interacting with hnRNP H1.


Assuntos
Proteínas do Capsídeo/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/fisiologia , Animais , Proteínas do Capsídeo/genética , Ciclo Celular , Linhagem Celular , Proliferação de Células , Embrião de Galinha , Fibroblastos/virologia , Expressão Gênica , Técnicas de Silenciamento de Genes/veterinária , Ribonucleoproteínas Nucleares Heterogêneas/genética , Técnicas do Sistema de Duplo-Híbrido , Replicação Viral
10.
DNA Repair (Amst) ; 106: 103180, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34298488

RESUMO

Since the early stages of the pandemic, hydroxychloroquine (HCQ), a widely used drug with good safety profile in clinic, has come to the forefront of research on drug repurposing for COVID-19 treatment/prevention. Despite the decades-long use of HCQ in the treatment of diseases, such as malaria and autoimmune disorders, the exact mechanisms of action of this drug are only beginning to be understood. To date, no data are available on the genotoxic potential of HCQ in vitro or in vivo. The present study is the first investigation of the DNA damaging- and mutagenic effects of HCQ in mammalian cells in vitro, at concentrations that are comparable to clinically achievable doses in patient populations. We demonstrate significant induction of a representative oxidative DNA damage (8-oxodG) in primary mouse embryonic fibroblasts (MEFs) treated with HCQ at 5 and 25 µM concentrations (P = 0.020 and P = 0.029, respectively), as determined by enzyme-linked immunosorbent assay. Furthermore, we show significant mutagenicity of HCQ, manifest as 2.2- and 1.8-fold increases in relative cII mutant frequency in primary and spontaneously immortalized Big Blue® MEFs, respectively, treated with 25 µM dose of this drug (P = 0.005 and P = 0.012, respectively). The observed genotoxic effects of HCQ in vitro, achievable at clinically relevant doses, are novel and important, and may have significant implications for safety monitoring in patient populations. Given the substantial number of the world's population receiving HCQ for the treatment of various chronic diseases or in the context of clinical trials for COVID-19, our findings warrant further investigations into the biological consequences of therapeutic/preventive use of this drug.


Assuntos
Hidroxicloroquina/farmacologia , Mutação/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Fibroblastos/efeitos dos fármacos , Fibroblastos/virologia , Mamíferos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Pandemias/prevenção & controle , SARS-CoV-2/efeitos dos fármacos
11.
Molecules ; 26(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299596

RESUMO

Zika virus (ZIKV) represents a re-emerging threat to global health due to its association with congenital birth defects. ZIKV NS2B-NS3 protease is crucial for virus replication by cleaving viral polyprotein at various junctions to release viral proteins and cause cytotoxic effects in ZIKV-infected cells. This study characterized the inhibitory effects of doxycycline against ZIKV NS2B-NS3 protease and viral replication in human skin cells. The in silico data showed that doxycycline binds to the active site of ZIKV protease at a low docking energy (-7.8 Kcal/mol) via four hydrogen bonds with the protease residues TYR1130, SER1135, GLY1151, and ASP83. Doxycycline efficiently inhibited viral NS2B-NS3 protease at average human temperature (37 °C) and human temperature with a high fever during virus infection (40 °C). Interestingly, doxycycline showed a higher inhibitory effect at 40 °C (IC50 = 5.3 µM) compared to 37 °C (9.9 µM). The virus replication was considerably reduced by increasing the concentration of doxycycline. An approximately 50% reduction in virus replication was observed at 20 µM of doxycycline. Treatment with 20 µM of doxycycline reduced the cytopathic effects (CPE), and the 40 µM of doxycycline almost eliminated the CPE of human skin cells. This study showed that doxycycline binds to the ZIKV protease and inhibits its catalytic activity at a low micro-molecular concentration range. Treatment of human skin fibroblast with doxycycline eliminated ZIKV infection and protected the cells against the cytopathic effects of the infection.


Assuntos
Doxiciclina/farmacologia , Fibroblastos/metabolismo , Pele/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Zika virus/fisiologia , Animais , Chlorocebus aethiops , Doxiciclina/química , Fibroblastos/virologia , Humanos , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Pele/virologia , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Zika virus/química
12.
Vet Microbiol ; 260: 109167, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34274763

RESUMO

Virulent Newcastle disease virus (NDV) is a violent infection in avian species. The understanding of its pathogenic mechanism is consistently evolving along with the development of molecular biological advancement. Exosomes derived from NDV infected cells (NDV Ex) were reported to promote virus replication through transportation of viral proteins and miRNAs. However, the function of mRNAs in NDV Ex remains unknown. In this study, a novel mechanism of NDV Ex to facilitate NDV infection was explored. Through transcriptome analysis, seven immune related genes were found to up-regulate in NDV Ex. Among them, NLRX1 mRNA was notably enriched in NDV Ex, and decreased inside the cells after virulent NDV infection. Further investigation suggested that NLRX1 mRNA decrease was in accordance with the NLRX1 protein expression reduction. This process can be reversed by the inhibition of exosome release. Therefore, NDV infection could utilize NDV Ex to export NLRX1 mRNA and reduce cellular NLRX1 protein. As NLRX1 is a crucial anti-viral protein of MAVS signal pathway, and NDV Ex transported NLRX1 cannot counteract its function in recipient cells, it can be concluded that NDV could benefit its replication through exporting NLRX1 mRNA to relieve the anti-viral pressure on its survival.


Assuntos
Exossomos/metabolismo , Proteínas Mitocondriais/metabolismo , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/patogenicidade , Transdução de Sinais , Transcriptoma , Proteínas Virais/metabolismo , Animais , Transporte Biológico , Embrião de Galinha , Fibroblastos/virologia , Células HeLa , Humanos , Proteínas Mitocondriais/genética , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/fisiologia , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Proteínas Virais/genética , Replicação Viral
13.
Med Microbiol Immunol ; 210(4): 197-209, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34091753

RESUMO

Polymorphonuclear leukocytes (PMNs) are regarded as vehicles for the hematogenous dissemination of human cytomegalovirus (HCMV). In cell culture, this concept has been validated with cell-free laboratory strains but not yet with clinical HCMV isolates that grow strictly cell-associated. We, therefore, aimed to evaluate whether PMNs can also transmit such isolates from initially infected fibroblasts to other cell types, which might further clarify the role of PMNs in HCMV dissemination and provide a model to search for potential inhibitors. PMNs, which have been isolated from HCMV-seronegative individuals, were added for 3 h to fibroblasts infected with recent cell-associated HCMV isolates, then removed and transferred to various recipient cell cultures. The transfer efficiency in the recipient cultures was evaluated by immunofluorescence staining of viral immediate early antigens. Soluble derivatives of the cellular HCMV entry receptor PDGFRα were analyzed for their potential to interfere with this transfer. All of five tested HCMV isolates could be transferred to fibroblasts, endothelial and epithelial cells with transfer rates ranging from 2 to 9%, and the transferred viruses could spread focally in these recipient cells within 1 week. The PDGFRα-derived peptides IK40 and GT40 reduced transfer by 40 and 70% when added during the uptake step. However, when added during the transfer step, only IK40 was effective, inhibiting transmission by 20% on endothelial cells and 50-60% on epithelial cells and fibroblasts. These findings further corroborate the assumption of cell-associated HCMV dissemination by PMNs and demonstrate that it is possible to inhibit this transmission mode.


Assuntos
Infecções por Citomegalovirus/transmissão , Infecções por Citomegalovirus/virologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/fisiologia , Neutrófilos/virologia , Peptídeos/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antígenos Virais/metabolismo , Antivirais/farmacologia , Linhagem Celular , Citomegalovirus/isolamento & purificação , Células Endoteliais/virologia , Células Epiteliais/virologia , Fibroblastos/virologia , Humanos , Peptídeos/química , Internalização do Vírus/efeitos dos fármacos
14.
J Virol ; 95(18): e0082121, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34191581

RESUMO

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. Distinct from other viruses studied to date, at mid- to late stages of its unusually protracted replication cycle, human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure, termed the assembly compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of transforming acidic coiled coil protein 3 (TACC3) to recruit the MT polymerase, chTOG, from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. IMPORTANCE Diverse viruses rely on host cell microtubule networks to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. However, precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the assembly compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid- to late stages of infection, HCMV increases the expression of transforming acidic coiled coil protein 3 (TACC3) to control the localization of the microtubule polymerase, chTOG. This, in turn, enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.


Assuntos
Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Fibroblastos/virologia , Complexo de Golgi/virologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Replicação Viral , Células Cultivadas , Derme/metabolismo , Derme/virologia , Fibroblastos/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/virologia
15.
Vet Microbiol ; 259: 109082, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34144834

RESUMO

MicroRNAs (miRNAs) are a class of ∼22 nucleotides non-coding RNAs that are encoded by a wide range of hosts. Viruses, especially herpesviruses, encode a variety of miRNAs that involved in disease progression. Recently, a cluster of virus-encoded miRNAs, miR-M8-M10, have been shown to restrict early cytolytic replication and pathogenesis of Marek's disease virus (MDV), an oncogenic avian alphaherpesvirus that causes lymphoproliferative disease in chickens. In this study, we specifically dissected the role of miR-M7, a member of cluster miR-M8-M10, in regulating MDV replication and pathogenesis. We found that deletion of miR-M7-5p did not affect the virus plaque size and growth in cell culture. However, compared to parental virus, infection of miR-M7-5p deletion virus significantly increased MDV genome copy number at 5 days post infection, suggesting that miR-M7 plays a role to restrict MDV replication during early cytolytic phase. In addition, our results showed that infection of miR-M7-5p deletion virus significantly enhanced the mortality of chickens, even it induced lymphoid organ atrophy similar to parental and revertant viruses. Taken together, our study revealed that the miR-M7 acts as a repressive factor of MDV replication and pathogenesis.


Assuntos
Herpesvirus Galináceo 2/genética , Herpesvirus Galináceo 2/patogenicidade , MicroRNAs/genética , Proteínas Virais/genética , Replicação Viral/genética , Animais , Células Cultivadas , Galinhas/virologia , Fibroblastos/virologia , Deleção de Genes , Herpesvirus Galináceo 2/crescimento & desenvolvimento , Doença de Marek/virologia , Organismos Livres de Patógenos Específicos , Fatores de Virulência/genética
16.
Nat Commun ; 12(1): 3910, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162877

RESUMO

Citrullination is the conversion of arginine-to-citrulline by protein arginine deiminases (PADs), whose dysregulation is implicated in the pathogenesis of various types of cancers and autoimmune diseases. Consistent with the ability of human cytomegalovirus (HCMV) to induce post-translational modifications of cellular proteins to gain a survival advantage, we show that HCMV infection of primary human fibroblasts triggers PAD-mediated citrullination of several host proteins, and that this activity promotes viral fitness. Citrullinome analysis reveals significant changes in deimination levels of both cellular and viral proteins, with interferon (IFN)-inducible protein IFIT1 being among the most heavily deiminated one. As genetic depletion of IFIT1 strongly enhances HCMV growth, and in vitro IFIT1 citrullination impairs its ability to bind to 5'-ppp-RNA, we propose that viral-induced IFIT1 citrullination is a mechanism of HCMV evasion from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection.


Assuntos
Citomegalovirus/metabolismo , Fibroblastos/metabolismo , Processamento de Proteína Pós-Traducional , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Cultivadas , Chlorocebus aethiops , Citrulinação , Citomegalovirus/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/virologia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Resistência a Myxovirus/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células Vero , Proteínas Virais/metabolismo
17.
Mol Cell ; 81(15): 3171-3186.e8, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34171297

RESUMO

Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that arginine monomethylation precisely regulates the mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. Protein arginine methyltransferase 7 (PRMT7) forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled in a timely manner due to automethylation at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression of MAVS activation. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo but also uncover a mechanism by which PRMT7 is tightly controlled to ensure the timely activation of antiviral defense.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Arginina/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Imunidade Inata/fisiologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Proteína DEAD-box 58/metabolismo , Fibroblastos/virologia , Células HEK293 , Herpes Simples/imunologia , Herpes Simples/metabolismo , Herpes Simples/virologia , Humanos , Metilação , Camundongos , Camundongos Knockout , Alcamidas Poli-Insaturadas , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/imunologia , Receptores Imunológicos/metabolismo , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/metabolismo , Infecções por Respirovirus/virologia , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
18.
Sci Rep ; 11(1): 11084, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34040106

RESUMO

Marek's disease virus (MDV) induces severe immunosuppression and lymphomagenesis in the chicken, its natural host, and results in a condition that investigated the pathogenesis of MDV and have begun to focus on the expression profiling of circular RNAs (circRNAs). However, little is known about how the expression of circRNAs is referred to as Marek's disease. Previous reports have is regulated during MDV replication. Here, we carried out a comprehensive profiling analysis of N6-methyladenosine (m6A) modification on the circRNA transcriptome in infected and uninfected chicken embryonic fibroblast (CEF) cells. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) revealed that m6A modification was highly conserved in circRNAs. Comparing to the uninfected group, the number of peaks and conserved motifs were not significantly different in cells that were infected with MDV, although reduced abundance of circRNA m6A modifications. However, gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses revealed that the insulin signaling pathway was associated with the regulation of m6A modified circRNAs in MDV infection. This is the first report to describe alterations in the transcriptome-wide profiling of m6A modified circRNAs in MDV-infected CEF cells.


Assuntos
Herpesvirus Galináceo 2/genética , Doença de Marek/virologia , RNA Circular/genética , Animais , Células Cultivadas , Galinhas , Fibroblastos/virologia , Perfilação da Expressão Gênica , Doença de Marek/genética
19.
Res Vet Sci ; 137: 68-76, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33933710

RESUMO

In this study, the effects of Goose parvovirus (GPV) infection as well as the possible role of NS1 protein on apoptosis induction in goose embryo fibroblast (GEF) cells were examined. Flow cytometry analysis and TUNEL assays revealed that GPV infection and NS1 transfection induced significant apoptosis in GEF cells compared to what was observed in mock-infected cells. Interestingly, the increase in the rate of apoptosis detected in GPV-infected GEFs was accompanied by an increased viral load in the cells. In addition, the apoptotic pathway was mediated by apoptosis-inducing factors (AIFs) and internal factors that influence the release of AIFs. The results indicated that the mitochondrial membrane potential was decreased, and AIF expression was increased in the nucleus (P < 0.01). Reactive oxygen species (ROS) increased gradually within 48 h (P < 0.001). Cathepsin D activities were also increased (P < 0.05). The results demonstrated that the AIF-mediated pathway is a new mitochondrial apoptotic pathway and that mitochondrial depolarization, ROS content, and cathepsin D activities are the key factors influencing apoptosis in GEF cells.


Assuntos
Fibroblastos/virologia , Gansos/embriologia , Parvovirinae/patogenicidade , Proteínas não Estruturais Virais/farmacologia , Animais , Apoptose , Fator de Indução de Apoptose/metabolismo , Fator de Indução de Apoptose/farmacologia , Catepsina D/metabolismo , Morte Celular , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo
20.
Cells ; 10(5)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946985

RESUMO

Human cytomegalovirus (HCMV) and Human herpesvirus 6 (HHV-6) have been reportedly suggested as triggers of the onset and/or progression of systemic sclerosis (SSc), a severe autoimmune disorder characterized by multi-organ fibrosis. The etiology and pathogenesis of SSc are still largely unknown but virological and immunological observations support a role for these beta-herpesviruses, and we recently observed a direct impact of HCMV and HHV-6 infection on the expression of cell factors associated with fibrosis at the cell level. Since miRNA expression has been found profoundly deregulated at the tissue level, here we aimed to investigate the impact on cell microRNome (miRNome) of HCMV and HHV-6 infection in in vitro infected primary human dermal fibroblasts, which represent one of the main SSc target cells. The analysis, performed by Taqman arrays detecting and quantifying 754 microRNAs (miRNAs), showed that both herpesviruses significantly modulated miRNA expression in infected cells, with evident early and late effects and deep modulation (>10 fold) of >40 miRNAs at each time post infection, including those previously recognized for their key function in fibrosis. The correlation between these in vitro results with in vivo observations is strongly suggestive of a role of HCMV and/or HHV-6 in the multistep pathogenesis of fibrosis in SSc and in the induction of fibrosis-signaling pathways finally leading to tissue fibrosis. The identification of specific miRNAs may open the way to their use as biomarkers for SSc diagnosis, assessment of disease progression and possible antifibrotic therapies.


Assuntos
Infecções por Citomegalovirus/genética , Fibroblastos/metabolismo , MicroRNAs/genética , Infecções por Roseolovirus/genética , Transcriptoma , Células Cultivadas , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/metabolismo , Fibroblastos/patologia , Fibroblastos/virologia , Fibrose , Herpesvirus Humano 6/patogenicidade , Humanos , MicroRNAs/metabolismo , Infecções por Roseolovirus/metabolismo , Escleroderma Sistêmico/etiologia
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