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1.
Vet Microbiol ; 239: 108460, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767079

RESUMO

Newcastle disease (ND) is an acute and contagious avian disease caused by Newcastle disease virus (NDV). MicroRNAs (miRNAs) play a significant role in host-pathogen interactions and the innate immune response. However, the role of miRNAs in the host response to NDV infection is not clearly understood. In this study, we showed that expression of the cellular miRNA gga-miR-455-5p was downregulated in vivo and in vitro in response to NDV infection. Next, we found that the transfection of chicken embryonic fibroblasts (CEFs) with gga-miR-455-5p suppressed NDV replication, while the blockade of endogenous gga-miR-455-5p expression with inhibitors enhanced NDV replication. In addition, gga-miR-455-5p enhanced the expression of type I interferon and the interferon-inducible genes (ISGs) OASL and Mx1 by targeting SOCS3, a negative regulator of type I IFN signaling. Altogether, these findings highlight the crucial role of gga-miR-455-5p in host defense against NDV by targeting the SOCS3 gene to inhibit NDV replication.


Assuntos
MicroRNAs/genética , MicroRNAs/metabolismo , Vírus da Doença de Newcastle/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Replicação Viral/genética , Animais , Células Cultivadas , Regulação para Baixo , Fibroblastos/virologia , Interações entre Hospedeiro e Microrganismos/genética , Interferon Tipo I/genética , Doença de Newcastle/fisiopatologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Transdução de Sinais/genética
2.
Nat Commun ; 10(1): 4878, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653857

RESUMO

Herpesvirus infection initiates a range of perturbations in the host cell, which remain poorly understood at the level of individual cells. Here, we quantify the transcriptome of single human primary fibroblasts during the first hours of lytic infection with HSV-1. By applying a generalizable analysis scheme, we define a precise temporal order of early viral gene expression and propose a set-wise emergence of viral genes. We identify host cell genes and pathways relevant for infection by combining three different computational approaches: gene and pathway overdispersion analysis, prediction of cell-state transition probabilities, as well as future cell states. One transcriptional program, which correlates with increased resistance to infection, implicates the transcription factor NRF2. Consequently, Bardoxolone methyl and Sulforaphane, two known NRF2 agonists, impair virus production, suggesting that NRF2 activation restricts viral infection. Our study provides insights into early stages of HSV-1 infection and serves as a general blueprint for the investigation of heterogeneous cell states in virus infection.


Assuntos
Fibroblastos/metabolismo , Herpes Simples/genética , Herpesvirus Humano 1 , Interações Hospedeiro-Patógeno/genética , Fator 2 Relacionado a NF-E2/genética , Fibroblastos/virologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Isotiocianatos/farmacologia , Fator 2 Relacionado a NF-E2/agonistas , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Cultura Primária de Células , Análise de Sequência de RNA , Análise de Célula Única , Replicação Viral/efeitos dos fármacos
3.
Vet Microbiol ; 237: 108381, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31585646

RESUMO

The H5N8 highly pathogenic avian influenza viruses (HPAIVs) isolated in Japan during the 2014-2015 winter differed in their pathogenicity in chickens. In the present study, we examined the possibility that a comparatively less pathogenic strain was first brought into the country by migratory birds, and then acquired enhanced pathogenicity by infecting chicken flocks. We showed that the A/tundra swan/Tottori/C6nk/2014 (H5N8) (Tottori P0) strain required 10 days to kill all chickens via the intranasal route. However, Tottori P1-B, a strain recovered from the brain of a chicken infected with parental Tottori P0, showed enhanced pathogenicity; Tottori P1-B replicated significantly in the lung and liver, and killed all infected birds within 6 days, which was comparable to a chicken farm isolate obtained in the same season, A/environment/Miyazaki/11/2014 (H5N8). Tottori P1-B showed more marked proliferation in MDCK and chicken fibroblast cells, especially during the early phase of infection. Sequence analysis revealed a single mutation, M374 V, in nucleoprotein (NP) of the passaged virus, and this substitution was conserved after a further inoculation study. Position 374 in NP is located in the functional domain interacting with polymerase protein, PB2, indicating that viral polymerase activity was involved in the rapid growth of Tottori P1-B in vitro and in vivo. These results suggest that HPAIV, which originally had comparatively low pathogenicity to chickens, can increase its pathogenicity through the infection from migratory birds to domestic chickens.


Assuntos
Galinhas , Patos , Vírus da Influenza A Subtipo H5N8/patogenicidade , Influenza Aviária/virologia , Animais , Linhagem Celular , Embrião de Galinha , Cães , Fibroblastos/virologia , Modelos Moleculares , Conformação Proteica , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência
4.
Arch Virol ; 164(12): 2995-3006, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31576460

RESUMO

Reticuloendotheliosis virus (REV) is an important representative avian retrovirus. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of REV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect changes in protein levels in chicken embryo fibroblast cells (CEFs) that were infected with REV or mock infected. In total, 605 cellular proteins were differentially expressed, among which 196, 345, and 286 were differentially expressed in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and immune system processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding, catalytic activity, and enzyme regulator activity. Pathway analysis showed that a total of 143, 167, and 179 pathways, including protein digestion and absorption, focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, Toll-like receptors, and JAK-STAT signaling, were enriched in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. In conclusion, this study is the first to analyze the protein profile of REV-infected CEFs using an iTRAQ approach. The results of this study provide valuable information for better understanding the host response to REV infection.


Assuntos
Fibroblastos/metabolismo , Doenças das Aves Domésticas/genética , Proteoma/genética , Vírus da Reticuloendoteliose/fisiologia , Infecções por Retroviridae/veterinária , Animais , Embrião de Galinha , Galinhas , Fibroblastos/química , Fibroblastos/virologia , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/virologia , Proteoma/química , Proteoma/metabolismo , Proteômica , Vírus da Reticuloendoteliose/genética , Infecções por Retroviridae/genética , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/virologia , Espectrometria de Massas em Tandem
5.
Nature ; 574(7777): 259-263, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554973

RESUMO

Chikungunya virus (CHIKV) is a re-emerging alphavirus that is transmitted to humans by mosquito bites and causes musculoskeletal and joint pain1,2. Despite intensive investigations, the human cellular factors that are critical for CHIKV infection remain unknown, hampering the understanding of viral pathogenesis and the development of anti-CHIKV therapies. Here we identified the four-and-a-half LIM domain protein 1 (FHL1)3 as a host factor that is required for CHIKV permissiveness and pathogenesis in humans and mice. Ablation of FHL1 expression results in the inhibition of infection by several CHIKV strains and o'nyong-nyong virus, but not by other alphaviruses and flaviviruses. Conversely, expression of FHL1 promotes CHIKV infection in cells that do not normally express it. FHL1 interacts directly with the hypervariable domain of the nsP3 protein of CHIKV and is essential for the replication of viral RNA. FHL1 is highly expressed in CHIKV-target cells and is particularly abundant in muscles3,4. Dermal fibroblasts and muscle cells derived from patients with Emery-Dreifuss muscular dystrophy that lack functional FHL15 are resistant to CHIKV infection. Furthermore,  CHIKV infection  is undetectable in Fhl1-knockout mice. Overall, this study shows that FHL1 is a key factor expressed by the host that enables CHIKV infection and identifies the interaction between nsP3 and FHL1 as a promising target for the development of anti-CHIKV therapies.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/patogenicidade , Fatores Celulares Derivados do Hospedeiro/metabolismo , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Animais , Células Cultivadas , Febre de Chikungunya/tratamento farmacológico , Vírus Chikungunya/efeitos dos fármacos , Vírus Chikungunya/genética , Vírus Chikungunya/crescimento & desenvolvimento , Feminino , Fibroblastos/virologia , Células HEK293 , Fatores Celulares Derivados do Hospedeiro/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/deficiência , Proteínas com Domínio LIM/genética , Masculino , Camundongos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Mioblastos/virologia , Vírus O'nyong-nyong/crescimento & desenvolvimento , Vírus O'nyong-nyong/patogenicidade , Ligação Proteica , RNA Viral/biossíntese , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral
6.
J Microbiol Biotechnol ; 29(11): 1790-1798, 2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31546296

RESUMO

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'- end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28°C but weak at 30°C. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.


Assuntos
Fibroblastos/virologia , Genes Reporter/genética , Nodaviridae/fisiologia , RNA Viral/metabolismo , Animais , Linhagem Celular , Cricetinae , Expressão Gênica , Genoma Viral/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Nodaviridae/genética , RNA Viral/genética , Tymovirus/genética , Replicação Viral
7.
Mol Biotechnol ; 61(10): 783-790, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31482466

RESUMO

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.


Assuntos
Flavivirus/metabolismo , Mardivirus/crescimento & desenvolvimento , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Cromossomos Artificiais Bacterianos/genética , Fibroblastos/citologia , Fibroblastos/virologia , Flavivirus/genética , Mardivirus/genética , Proteínas do Envelope Viral/genética , Vacinas Virais/metabolismo
8.
Mol Cell ; 76(3): 485-499.e8, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31495563

RESUMO

Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to "reprogram" cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.


Assuntos
Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fibroblastos/efeitos dos fármacos , Interferon gama/farmacologia , Transcrição Genética/efeitos dos fármacos , Animais , Quinase 8 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Fibroblastos/enzimologia , Fibroblastos/virologia , Células HCT116 , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , RNA Polimerase II/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Vesiculovirus/patogenicidade
9.
Adv Virus Res ; 104: 283-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31439151

RESUMO

In this chapter, we present an overview on betaherpesvirus entry, with a focus on human cytomegalovirus, human herpesvirus 6A and human herpesvirus 6B. Human cytomegalovirus (HCMV) is a complex human pathogen with a genome of 235kb encoding more than 200 genes. It infects a broad range of cell types by switching its viral ligand on the virion, using the trimer gH/gL/gO for infection of fibroblasts and the pentamer gH/gL/UL128/UL130/UL131 for infection of other cells such as epithelial and endothelial cells, leading to membrane fusion mediated by the fusion protein gB. Adding to this scenario, however, accumulating data reveal the actual complexity in the viral entry process of HCMV with an intricate interplay among viral and host factors. Key novel findings include the identification of entry receptors platelet-derived growth factor-α receptor (PDGFRα) and Netropilin-2 (Nrp2) for trimer and pentamer, respectively, the determination of atomic structures of the fusion protein gB and the pentamer, and the in situ visualization of the state and arrangement of functional glycoproteins on virion. This is covered in the first part of this review. The second part focusses on HHV-6 which is a T lymphotropic virus categorized as two distinct virus species, HHV-6A and HHV-6B based on differences in epidemiological, biological, and immunological aspects, although homology of their entire genome sequences is nearly 90%. HHV-6B is a causative agent of exanthema subitum (ES), but the role of HHV-6A is unknown. HHV-6B reactivation occasionally causes encephalitis in patients with hematopoietic stem cell transplant. The HHV-6 specific envelope glycoprotein complex, gH/gL/gQ1/gQ2 is a viral ligand for the entry receptor. Recently, each virus has been found to recognize a different cellular receptor, CD46 for HHV 6A amd CD134 for HHV 6B. These findings show that distinct receptor recognition differing between both viruses could explain their different pathogenesis.


Assuntos
Citomegalovirus/fisiologia , Herpesvirus Humano 6/fisiologia , Internalização do Vírus , Células Endoteliais/virologia , Células Epiteliais/virologia , Fibroblastos/virologia , Glicoproteínas/metabolismo , Humanos , Proteína Cofatora de Membrana/metabolismo , Neuropilina-2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores OX40/metabolismo , Receptores Virais/metabolismo , Linfócitos T/virologia , Proteínas do Envelope Viral/metabolismo
10.
PLoS Pathog ; 15(8): e1008002, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31404116

RESUMO

The galectin 3 binding protein (LGALS3BP, also known as 90K) is a ubiquitous multifunctional secreted glycoprotein originally identified in cancer progression. It remains unclear how 90K functions in innate immunity during viral infections. In this study, we found that viral infections resulted in elevated levels of 90K. Further studies demonstrated that 90K expression suppressed virus replication by inducing IFN and pro-inflammatory cytokine production. Upon investigating the mechanisms behind this event, we found that 90K functions as a scaffold/adaptor protein to interact with TRAF6, TRAF3, TAK1 and TBK1. Furthermore, 90K enhanced TRAF6 and TRAF3 ubiquitination and served as a specific ubiquitination substrate of TRAF6, leading to transcription factor NF-κB, IRF3 and IRF7 translocation from the cytoplasm to the nucleus. Conclusions: 90K is a virus-induced protein capable of binding with the TRAF6 and TRAF3 complex, leading to IFN and pro-inflammatory production.


Assuntos
Antígenos de Neoplasias/fisiologia , Biomarcadores Tumorais/fisiologia , Glicoproteínas/fisiologia , Fator 3 Associado a Receptor de TNF/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/antagonistas & inibidores , Viroses/imunologia , Replicação Viral , Vírus/imunologia , Animais , Células Cultivadas , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Viroses/metabolismo , Viroses/virologia
11.
Appl Microbiol Biotechnol ; 103(20): 8473-8483, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31468087

RESUMO

Type III interferon (IFN-λ) has recently been shown to exert a significant antiviral impact against viruses in vertebrates. Avian leukosis virus subgroup J (ALV-J), which causes tumor disease and immunosuppression in infected chicken, is a retrovirus that is difficult to prevent and control because of a lack of vaccines and drugs. Here, we obtained chicken IFN-λ (chIFN-λ) using a silkworm bioreactor and demonstrated that chIFN-λ has antiviral activity against ALV-J infection of both chicken embryo fibroblast cell line (DF1) and epithelial cell line (LMH). We found that chIFN-λ triggered higher levels of particular type III interferon-stimulated genes (type III ISGs) including myxovirus resistance protein (Mx), viperin (RSAD2), and interferon-inducible transmembrane protein 3 (IFITM3) in DF1 and LMH cells. Furthermore, over-expression of Mx, viperin, and IFITM3 could inhibit ALV-J infection in DF1 and LMH cells. Therefore, these results suggested that the anti-ALV-J function of chIFN-λ was specifically implemented by induction of expression of type III ISGs. Our data identified chIFN-λ as a critical antiviral agent of ALV-J infection and provides a potentially and attractive platform for the production of commercial chIFN-λ.


Assuntos
Antivirais/metabolismo , Vírus da Leucose Aviária/crescimento & desenvolvimento , Galinhas , Interferons/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Reatores Biológicos , Bombyx , Células Epiteliais/virologia , Fibroblastos/virologia , Expressão Gênica , Interferons/genética , Proteínas Recombinantes/genética
12.
PLoS Pathog ; 15(8): e1007993, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31465513

RESUMO

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3'-Cre). CHIKV-3'-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3'-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3'-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease.


Assuntos
Artrite Experimental/virologia , Febre de Chikungunya/virologia , Vírus Chikungunya/patogenicidade , Derme/patologia , Fibroblastos/patologia , Músculo Esquelético/patologia , RNA Viral/metabolismo , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Febre de Chikungunya/metabolismo , Vírus Chikungunya/genética , Derme/metabolismo , Derme/virologia , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/virologia , Músculo Esquelético/metabolismo , Músculo Esquelético/virologia , RNA Viral/genética , Replicação Viral
13.
Nature ; 571(7765): 419-423, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292545

RESUMO

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.


Assuntos
Regulação da Expressão Gênica/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única , Transcrição Genética/genética , Alquilação , Animais , Ciclo Celular , Citomegalovirus/fisiologia , Metilação de DNA , Fibroblastos/metabolismo , Fibroblastos/virologia , Cinética , Camundongos , Regiões Promotoras Genéticas/genética , RNA/análise , RNA/química , Compostos de Sulfidrila/química
14.
Vet Microbiol ; 235: 151-163, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282373

RESUMO

This study demonstrates that the Muscovy duck reovirus (MDRV) p10.8 protein is one of many viral non-structural proteins that induces both cell cycle arrest and apoptosis. The p10.8 but not σC is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. Our results reveal that p10.8-induced apoptosis in cultured cells occurs by the nucleoporin Tpr/p53-dependent and Fas/caspase 8-mediated pathways. Furthermore, a compelling finding from this study is that the p10.8 and σC proteins of MDRV facilitate CDK2 and CDK4 degradation via the ubiquitin-proteasome pathway. We found that depletion of Cdc20 reversed the p10.8- and σC- mediated CDK4 degradation and p10.8-induced apoptosis, suggesting that Cdc20 plays a critical role in modulating p10.8-mediated cell cycle and apoptosis. Furthermore, we found that depletion of chaperonin-containing tailless complex polypeptide 1 (CCT) 2 and CCT5 reduced the level of Cdc20 and reversed the p10.8- and σC-mediated CDK4 degradation and p10.8-induced apoptosis, indicating that molecular chaperone CCT2 and CCT5 are required for stabilization of Ccd20 for mediating both cell cycle arrest and apoptosis. This study provides mechanistic insights into how p10.8 induces both cell cycle arrest and apoptosis.


Assuntos
Proteínas Cdc20/metabolismo , Chaperonina com TCP-1/metabolismo , Orthoreovirus/genética , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/veterinária , Proteínas não Estruturais Virais/metabolismo , Animais , Apoptose , Caspase 8/genética , Caspase 8/metabolismo , Proteínas Cdc20/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Chaperonina com TCP-1/genética , Patos/virologia , Fibroblastos/virologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , RNA Interferente Pequeno , Células Vero , Proteínas não Estruturais Virais/genética
15.
Poult Sci ; 98(11): 6019-6025, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31309233

RESUMO

Emerging evidence suggests that some members of the tripartite motif (TRIM) family play a crucial role in antiretroviral. However, the chicken TRIM62 antiretroviral activity is unknown. Avian leukosis virus subgroup J (ALV-J) is an avian retrovirus mainly inducing tumor formation and immunosuppression. The purpose of the study was to explore chicken TRIM62's role in ALV-J replication. In this study, we first tested the RNA expression of ALV-J and TRIM62 in chicken embryo fibroblasts (CEFs) cells infected with ALV-J by qRT-PCR. The result showed that ALV-J infection affected TRIM62 RNA expression, first upregulation and then downregulation, with the time course infection of ALV-J. Then, we silenced and overexpressed the TRIM62 to evaluate the effect of TRIM62 on ALV-J replication by qRT-PCR. We found that the knockdown of TRIM62 in CEF cells with shRNA targeting SPRY domain enhanced the viral replication more significantly than that with shRNA targeting coiled coil/unstructured domain, and overexpression of TRIM62 inhibited the viral replication. Further, we detected the effect of the domain deletion on TRIM62's antiviral activity. The result demonstrated that deletion of RING, B-box, coiled-coil domains partially abolished TRIM62's antiviral activity, while SPRY domain deletion resulted in the disappearance of antiviral activity of TRIM62. Taken together, our findings strongly suggested that TRIM62 plays an important role in the restriction of ALV-J replication, and SPRY domain is a prerequisite for the antiviral activity of TRIM62.


Assuntos
Vírus da Leucose Aviária/fisiologia , Proteínas Aviárias/metabolismo , Domínio B30.2-SPRY/genética , Galinhas/virologia , Proteínas com Motivo Tripartido/metabolismo , Replicação Viral , Animais , Embrião de Galinha , Fibroblastos/virologia , Regulação da Expressão Gênica
16.
PLoS Pathog ; 15(7): e1007914, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31356650

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause disability in newborns and serious clinical diseases in immunocompromised patients. HCMV has a large genome with enormous coding potential; its viral particles are equipped with complicated glycoprotein complexes and can infect a wide range of human cells. Although multiple host cellular receptors interacting with viral glycoproteins have been reported, the mechanism of HCMV infection remains a mystery. Here we report identification of adipocyte plasma membrane-associated protein (APMAP) as a novel modulator active in the early stage of HCMV infection. APMAP is necessary for HCMV infection in both epithelial cells and fibroblasts; knockdown of APMAP expression significantly reduced HCMV infection of these cells. Interestingly, ectopic expression of human APMAP in cells refractory to HCMV infection, such as canine MDCK and murine NIH/3T3 cells, promoted HCMV infection. Furthermore, reduction in viral immediate early (IE) gene transcription at 6 h post infection and delayed nucleus translocation of tegument delivered pp65 at 4 h post infection were detected in APMAP-deficient cells but not in the wildtype cells. These results suggest that APMAP plays a role in the early stage of HCMV infection. Results from biochemical studies of APMAP and HCMV proteins suggest that APMAP could participate in HCMV infection through interaction with gH/gL containing glycoprotein complexes at low pH and mediate nucleus translocation of tegument pp65. Taken together, our results suggest that APMAP functions as a modulator promoting HCMV infection in multiple cell types and is an important player in the complex HCMV infection mechanism.


Assuntos
Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/patogenicidade , Glicoproteínas de Membrana/metabolismo , Adipócitos/metabolismo , Adipócitos/virologia , Animais , Membrana Celular/metabolismo , Membrana Celular/virologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/etiologia , Cães , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Técnicas de Inativação de Genes , Interações entre Hospedeiro e Microrganismos , Humanos , Células Madin Darby de Rim Canino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Camundongos , Células NIH 3T3 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Estruturais Virais/metabolismo , Virulência , Internalização do Vírus
17.
PLoS Biol ; 17(7): e3000072, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31306410

RESUMO

Lymphoid T-zone fibroblastic reticular cells (FRCs) actively promote T-cell trafficking, homeostasis, and expansion but can also attenuate excessive T-cell responses via inducible nitric oxide (NO) and constitutive prostanoid release. It remains unclear how these FRC-derived mediators dampen T-cell responses and whether this occurs in vivo. Here, we confirm that murine lymph node (LN) FRCs produce prostaglandin E2 (PGE2) in a cyclooxygenase-2 (COX2)-dependent and inflammation-independent fashion. We show that this COX2/PGE2 pathway is active during both strong and weak T-cell responses, in contrast to NO, which only comes into play during strong T-cell responses. During chronic infections in vivo, PGE2-receptor signaling in virus-specific cluster of differentiation (CD)8 cytotoxic T cells was shown by others to suppress T-cell survival and function. Using COX2flox/flox mice crossed to mice expressing Cre recombinase expression under control of the CC chemokine ligand (CCL19) promoter (CCL19cre), we now identify CCL19+ FRC as the critical source of this COX2-dependent suppressive factor, suggesting PGE2-expressing FRCs within lymphoid tissues are an interesting therapeutic target to improve T-cell-mediated pathogen control during chronic infection.


Assuntos
Ciclo-Oxigenase 2/imunologia , Fibroblastos/imunologia , Linfonodos/imunologia , Prostaglandinas/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Movimento Celular/genética , Movimento Celular/imunologia , Proliferação de Células/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Linfonodos/citologia , Linfonodos/metabolismo , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Prostaglandinas/biossíntese , Linfócitos T/virologia
18.
Nat Commun ; 10(1): 2699, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221976

RESUMO

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.


Assuntos
Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Interações Hospedeiro-Patógeno/imunologia , Proteína Cofatora de Membrana/imunologia , Internalização do Vírus , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/administração & dosagem , Anticorpos Antivirais/imunologia , Linhagem Celular , Infecções por Citomegalovirus/prevenção & controle , Infecções por Citomegalovirus/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Técnicas de Inativação de Genes , Humanos , Proteína Cofatora de Membrana/genética , RNA Interferente Pequeno/metabolismo , Trofoblastos/imunologia , Trofoblastos/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia
19.
PLoS Negl Trop Dis ; 13(6): e0007537, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31251739

RESUMO

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.


Assuntos
Fatores Imunológicos/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Zika virus/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fibroblastos/imunologia , Fibroblastos/virologia , Humanos , Neurônios/imunologia , Neurônios/virologia , Ligação Proteica , Replicação Viral
20.
Vet Microbiol ; 233: 85-92, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176417

RESUMO

Muscovy duck reovirus (MDRV) causes serious immunodeficiency in the intestinal mucosa, although the underlying histopathological mechanisms remain unclear. Thus, we investigated the impact of MDRV infection on intestinal morphology using hematoxylin and eosin staining. Immune-related cells were also quantified by staining with hematoxylin and eosin, toluidine blue, and periodic acid-Schiff stain, or by immunohistochemistry and cytochemistry for lectin. Similarly, CD4+ and CD8+ cells were quantified by flow cytometry, and the expression of several immune-related molecules was quantified by radioimmunoassay. We found that MDRV clearly damaged the intestinal mucosa, based on tissue morphology, villus length, villus width, intestinal thickness, villus height/crypt depth ratio, and villus surface area. MDRV also altered the density or distribution of lymphocytes, mastocytes, and goblet cells in the small intestinal mucosa, as well as microfold cells in Peyer's patches. In addition, MDRV markedly depleted CD4+ cells from the intestinal mucosa and lowered the CD4+:CD8+ ratio in peripheral blood. Moreover, MDRV diminished the levels of secretory IgA and mucosal addressin cell adhesion molecule-1 (p < 0.01), but elevated those of histamine and nitric oxide (p < 0.01 or p < 0.05). Finally, MDRV significantly suppressed IL-1ß, IL-4, IL-5, and IL-8 levels (p < 0.01 or p < 0.05) mid-infection. Collectively, our data suggest that MDRV severely damages the structure and function of the intestinal mucosa by modulating immune cells and immune-related factors, thus leading to local immunodeficiency. Our findings lay the foundation for further research on the pathogenesis of MDRV.


Assuntos
Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/virologia , Orthoreovirus Aviário/imunologia , Infecções por Reoviridae/imunologia , Fatores Etários , Animais , Contagem de Linfócito CD4 , Citocinas/imunologia , Patos/virologia , Duodeno , Fibroblastos/virologia , Histamina/análise , Imunoglobulina A Secretora/análise , Intestino Delgado/imunologia , Óxido Nítrico/análise , Orthoreovirus Aviário/patogenicidade , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/patologia , Carga Viral
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