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Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious disease in chickens and seriously endangers the poultry industry. The emergence and co-circulation of diverse IBV serotypes and genotypes with distinct pathogenicity worldwide pose a serious challenge to the development of effective intervention measures. In this study, we report the epidemic trends of IBV in China from 2019 to 2023 and a comparative analysis on the antigenic characteristics and pathogenicity of isolates among major prevalent lineages. Phylogenetic and recombination analyses based on the nucleotide sequences of the spike (S) 1 gene clustered a total of 205 isolates into twelve distinct lineages, with GI-19 as a predominant lineage (61.77 ± 4.56%) exhibiting an overall increasing trend over the past five years, and demonstrated that a majority of the variants were derived from gene recombination events. Further characterization of the growth and pathogenic properties of six representative isolates from different lineages classified four out of the six isolates as nephropathogenic types with mortality rates in one-day-old SPF chickens varying from 20-60%, one as a respiratory type with weak virulence, and one as a naturally occurring avirulent strain. Taken together, our findings illuminate the epidemic trends, prevalence, recombination, and pathogenicity of current IBV strains in China, providing key information for further strengthening the surveillance and pathogenicity studies of IBV.
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Galinhas , Infecções por Coronavirus , Variação Genética , Genótipo , Vírus da Bronquite Infecciosa , Filogenia , Doenças das Aves Domésticas , Animais , Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/patogenicidade , Vírus da Bronquite Infecciosa/classificação , Vírus da Bronquite Infecciosa/isolamento & purificação , China/epidemiologia , Doenças das Aves Domésticas/virologia , Doenças das Aves Domésticas/epidemiologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Infecções por Coronavirus/epidemiologia , Prevalência , Virulência , Recombinação Genética , SorogrupoRESUMO
Introduction: Glucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms. Methods: The method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle. Results: Our study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication. Discussion: These findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.
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Chaperona BiP do Retículo Endoplasmático , Vírus da Doença de Newcastle , Animais , Neuraminidase/metabolismo , Hemaglutininas , Ligação Viral , Proteína HN/genética , Proteína HN/metabolismo , Proteína HN/farmacologia , Proteínas Virais/farmacologiaRESUMO
African swine fever virus (ASFV) causes a devastating viral hemorrhagic disease in domestic pigs and Eurasian wild boars, posing a foremost threat to the swine industry and pig farming. The development of an effective vaccine is urgently needed, but has been hampered by the lack of an in-depth, mechanistic understanding of the host immune response to ASFV infection and the induction of protective immunity. In this study, we report that immunization of pigs with Semliki Forest Virus (SFV) replicon-based vaccine candidates expressing ASFV p30, p54, and CD2v, as well as their ubiquitin-fused derivatives, elicits T cell differentiation and expansion, promoting specific T cell and humoral immunity. Due to significant variations in the individual non-inbred pigs in response to the vaccination, a personalized analysis was conducted. Using integrated analysis of differentially expressed genes (DEGs), Venn, KEGG and WGCNA, Toll-like receptor, C-type lectin receptor, IL17 receptor, NOD-like receptor and nucleic acid sensor-mediated signaling pathways were demonstrated to be positively correlated to the antigen-stimulated antibody production and inversely correlated to the IFN-γ secreting cell counts in peripheral blood mononuclear cells (PBMCs). An up-regulation of CIQA, CIQB, CIQC, C4BPA, SOSC3, S100A8 and S100A9, and down-regulation of CTLA4, CXCL2, CXCL8, FOS, RGS1, EGR1 and SNAI1 are general in the innate immune response post-the second boost. This study reveals that pattern recognition receptors TLR4, DHX58/DDX58 and ZBP1, and chemokines CXCL2, CXCL8 and CXCL10 may play important roles in regulating this vaccination-stimulated adaptive immune response.
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Vírus da Febre Suína Africana , Suínos , Animais , Vírus da Febre Suína Africana/genética , Vírus da Floresta de Semliki , Imunidade Humoral , Leucócitos Mononucleares , Sus scrofaRESUMO
Newcastle disease (ND) and infectious bursal disease (IBD) are two key infectious diseases that significantly threaten the health of the poultry industry. Although existing vaccinations can effectively prevent and treat these two diseases through multiple immunizations, frequent immunization stresses significantly impact chicken growth. In this study, three recombinant adenoviruses, rAd5-F expressing the NDV (genotype VII) F protein, rAd5-VP2 expressing the IBDV VP2 protein, and rAd5-VP2-F2A-F co-expressing F and VP2 proteins, were constructed using the AdEasy system. The F and VP2 genes of the recombinant adenoviruses could be transcribed and expressed normally in HEK293A cells as verified by RT-PCR and Western blot. The three recombinant viruses were shown to have similar growth kinetics as rAd5-EGFP. Compared with the PBS and rAd5-EGFP groups, SPF chickens immunized with recombinant adenoviruses produced higher antibody levels, more significant lymphocyte proliferation, and significantly higher CD4+/CD3+ and CD8+/CD3+ cells in peripheral blood. The survival rate of SPF chickens immunized with rAd5-F and rAd5-VP2-F2A-F after the challenge with DHN3 was 100%, and 86% of SPF chickens showed no viral shedding at 7 dpc. The survival rate of SPF chickens immunized with rAd5-VP2 and rAd5-VP2-F2A-F after the challenge with BC6/85 was 86%. rAd5-VP2 and rAd5-VP2-F2A-F significantly inhibited bursal atrophy and pathological changes compared to the rAd5-EGFP and PBS groups. This study provides evidence that these recombinant adenoviruses have the potential to be developed into safe and effective vaccine candidates for the prevention and control of ND and IBD.
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For industrial vaccine production, overwhelming the existing antiviral innate immune response dominated by type I interferons (IFN-I) in cells would be a key factor improving the effectiveness and production cost of vaccines. In this study, we report the construction of an IFN-I receptor 1 (IFNAR1)-knockout DF-1 cell line (KO-IFNAR1), which supports much more efficient replication of the duck Tembusu virus (DTMUV), Newcastle disease virus (NDV) and gammacoronavirus infectious bronchitis virus (IBV). Transcriptomic analysis of DTMUV-infected KO-IFNAR1 cells demonstrated that DTMUV mainly activated genes and signaling pathways related to cell growth and apoptosis. Among them, JUN, MYC and NFKBIA were significantly up-regulated. Furthermore, knockdown of zinc-fingered helicase 2 (HELZ2) and interferon-α-inducible protein 6 (IFI6), the two genes up-regulated in both wild type and KO-IFNAR1 cells, significantly increased the replication of DTMUV RNA. This study paves the way for further studying the mechanism underlying the DTMUV-mediated IFN-I-independent regulation of virus replication, and meanwhile provides a potential cell resource for efficient production of cell-based avian virus vaccines.
Assuntos
Infecções por Flavivirus , Flavivirus , Interferon Tipo I , Doenças das Aves Domésticas , Animais , Patos , Galinhas/genética , Transcriptoma , Flavivirus/genética , Linhagem Celular , Interferon Tipo I/genética , Antivirais , Apoptose , RNA , Interferon-alfa/genética , ZincoRESUMO
Coronavirus nonstructural protein 3 (nsp3) is a multi-functional protein, playing a critical role in viral replication and in regulating host antiviral innate immunity. In this study, we demonstrate that nsp3 from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and avian coronavirus infectious bronchitis virus (IBV) directly interacts with melanoma differentiation-associated gene 5 (MDA5), rendering an inhibitory effect on the MDA5-mediated type I interferon (IFN) response. By the co-expression of MDA5 with wild-type and truncated nsp3 constructs, at least three interacting regions mapped to the papain-like protease (PLpro) domain and two other domains located at the N- and C-terminal regions were identified in SARS-CoV-2 nsp3. Furthermore, by introducing point mutations to the catalytic triad, the deubiquitylation activity of the PLpro domain from both SARS-CoV-2 and IBV nsp3 was shown to be responsible for the suppression of the MDA5-mediated type I IFN response. It was also demonstrated that both MDA5 and nsp3 were able to interact with ubiquitin and ubiquitinated proteins, contributing to the interaction between the two proteins. This study confirms the antagonistic role of nsp3 in the MDA5-mediated type I IFN signaling, highlighting the complex interaction between a multi-functional viral protein and the innate immune response.
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Infecções por Coronavirus , Vírus da Bronquite Infecciosa , Interferon Tipo I , Helicase IFIH1 Induzida por Interferon , SARS-CoV-2 , Proteínas não Estruturais Virais , COVID-19 , Infecções por Coronavirus/imunologia , Humanos , Vírus da Bronquite Infecciosa/metabolismo , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , SARS-CoV-2/metabolismo , Ubiquitina/metabolismo , Proteínas Ubiquitinadas , Proteínas não Estruturais Virais/metabolismoRESUMO
Avian interferon regulatory factors 1 and 7 (IRF1 and IRF7) play important roles in the host's innate immunity against viral infection. Our previous study revealed that duck tembusu virus (DTMUV) infection of chicken fibroblasts (DF1) and duck embryo fibroblasts (DEFs) induced the expression of a variety of IFN-stimulated genes (ISGs), including VIPERIN, IFIT5, CMPK2, IRF1, and IRF7. IRF1 was further shown to play a significant role in regulating the up-expression of VIPERIN, IFIT5, and CMPK2 and inhibiting DTMUV replication. In this study, we confirm, through overexpression and knockout approaches, that both IRF1 and IRF7 inhibit DTMUV replication, mainly via regulation of type I IFN expression, as well as the induction of IRF1, VIPERIN, IFIT5, CMPK2, and MX1. In addition, IRF1 directly promoted the expression of VIPERIN and CMPK2 in an IFN-independent manner when IRF7 and type I IFN signaling were undermined. We also found that non-structural protein 2B (NS2B) of DTMUV was able to inhibit the induction of IFN-ß mRNA triggered by Newcastle disease virus (NDV) infection or poly(I:C) treatment, revealing a strategy employed by DTMUV to evade host's immunosurveillance. This study demonstrates that avian IRF7 and IRF1 play distinct roles in the regulation of type I IFN response during DTMUV infection.
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Patos , Flavivirus , Animais , Antivirais , Imunidade Inata , Interferon beta/genética , Vírus da Doença de NewcastleRESUMO
Low pathogenic avian influenza A(H9N2) virus is endemic worldwide and continually recruit internal genes to generate human-infecting H5N1, H5N6, H7N9, and H10N8 influenza variants. Here we show that hemagglutinin cleavage sites (HACS) of H9N2 viruses tended to mutate towards hydrophilic via evolutionary transition, and the tribasic HACS were found at high prevalence in Asia and the Middle East. Our finding suggested that the tribasic H9N2 viruses increased the viral replication, stability, pathogenicity and transmission in chickens and the virulence of mice compared to the monobasic H9N2 viruses. Notably, the enlarged stem-loop structures of HACS in the RNA region were found in the increasing tribasic H9N2 viruses. The enlarged HACS RNA secondary structures of H9N2 viruses did not influence the viral replication but accelerated the frequency of nucleotide insertion in HACS. With the prevailing tendency of the tribasic H9N2 viruses, the tribasic HACS in H9N2 viruses should be paid more attention.
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Evolução Molecular , Vírus da Influenza A Subtipo H9N2/genética , Animais , Galinhas , Hemaglutininas , Humanos , Vírus da Influenza A Subtipo H9N2/patogenicidade , Camundongos , Mutação , Filogeografia , Replicação ViralRESUMO
Elucidating virus-cell interactions is fundamental to understanding viral replication and identifying targets for therapeutic control of viral infection. The extracellular signal-regulated kinase (ERK) pathway has been shown to regulate pathogenesis during many viral infections, but its role during coronavirus infection is undetermined. Infectious bronchitis virus is the representative strain of Gammacoronavirus, which causes acute and highly contagious diseases in the poultry farm. In this study, we investigated the role of ERK1/2 signaling pathway in IBV infection. We found that IBV infection activated ERK1/2 signaling and the up-regulation of phosphatase DUSP6 formed a negative regulation loop. Pharmacological inhibition of MEK1/2-ERK1/2 signaling suppressed the expression of DUSP6, promoted cell death, and restricted virus replication. In contrast, suppression of DUSP6 by chemical inhibitor or siRNA increased the phosphorylation of ERK1/2, protected cells from apoptosis, and facilitated IBV replication. Overexpression of DUSP6 decreased the level of phospho-ERK1/2, promoted apoptosis, while dominant negative mutant DUSP6-DN lost the regulation function on ERK1/2 signaling and apoptosis. In conclusion, these data suggest that MEK-ERK1/2 signaling pathway facilitates IBV infection, probably by promoting cell survival; meanwhile, induction of DUSP6 forms a negative regulation loop to restrict ERK1/2 signaling, correlated with increased apoptosis and reduced viral load. Consequently, components of the ERK pathway, such as MEK1/2 and DUSP6, represent excellent targets for the development of antiviral drugs.
Assuntos
Apoptose/fisiologia , Fosfatases de Especificidade Dupla/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Vírus da Bronquite Infecciosa/fisiologia , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Animais , Butadienos/farmacologia , Linhagem Celular , Galinhas , Chlorocebus aethiops , Fosfatases de Especificidade Dupla/antagonistas & inibidores , Fosfatases de Especificidade Dupla/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Fosfatases da Proteína Quinase Ativada por Mitógeno/antagonistas & inibidores , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Nitrilas/farmacologia , Regulação para Cima , Replicação ViralRESUMO
Traditional Chinese medicine (TCM) has become popular and been viewed as an effective clinical treatment across the world. Accordingly, there is an ever-increasing interest in performing data analysis over TCM data. Aiming to cope with the problem of excessively depending on empirical values when selecting cluster centers by traditional clustering algorithms, an improved artificial bee colony algorithm is proposed by which to automatically select cluster centers and apply it to aggregate Chinese herbal medicines. The proposed method integrates the following new techniques: (1) improving the artificial bee colony algorithm by applying a new searching strategy of neighbour nectar, (2) employing the improved artificial bee colony algorithm to optimize the parameters of the cutoff distance dc, the local density ρi and the minimum distance δi between the element i and any other element with higher density in the cluster algorithm by fast search and finding of density peaks (called DP algorithm) to find the optimal cluster centers, in order to clustering herbal medicines in an accurate fashion with the guarantee of runtime performance. Extensive experiments were conducted on the UCI benchmark datasets and the TCM datasets and the results verify the effectiveness of the proposed method by comparing it with classical clustering algorithms including K-means, K-mediods and DBSCAN in multiple evaluation metrics, that is, Silhouette Coefficient, Entropy, Purity, Precision, Recall and F1-Measure. The results show that the IABC-DP algorithm outperforms other approaches with good clustering quality and accuracy on the UCI and the TCM datasets as well. In addition, it can be found that the improved artificial bee colony algorithm can effectively reduce the number of iterations when compared to the traditional bee colony algorithm. In particular, the IABC-DP algorithm is applied to cluster multi-dimensional Chinese herbal medicines and the result shows that it outperforms other clustering algorithms in clustering Chinese herbal medicines, which can contribute to a larger effort targeted at advancing the study of discovering composition rules of traditional Chinese prescriptions.
Assuntos
Algoritmos , Medicamentos de Ervas Chinesas , Análise por Conglomerados , HumanosRESUMO
BACKGROUND: Imbalanced datasets are commonly encountered in bioinformatics classification problems, that is, the number of negative samples is much larger than that of positive samples. Particularly, the data imbalance phenomena will make us underestimate the performance of the minority class of positive samples. Therefore, how to balance the bioinformatic data becomes a very challenging and difficult problem. RESULTS: In this study, we propose a new data sampling approach, called pseudo-negative sampling, which can be effectively applied to handle the case that: negative samples greatly dominate positive samples. Specifically, we design a supervised learning method based on a max-relevance min-redundancy criterion beyond Pearson correlation coefficient (MMPCC), which is used to choose pseudo-negative samples from the negative samples and view them as positive samples. In addition, MMPCC uses an incremental searching technique to select optimal pseudo-negative samples to reduce the computation cost. Consequently, the discovered pseudo-negative samples have strong relevance to positive samples and less redundancy to negative ones. CONCLUSIONS: To validate the performance of our method, we conduct experiments base on four UCI datasets and three real bioinformatics datasets. According to the experimental results, we clearly observe the performance of MMPCC is better than other sampling methods in terms of Sensitivity, Specificity, Accuracy and the Mathew's Correlation Coefficient. This reveals that the pseudo-negative samples are particularly helpful to solve the imbalance dataset problem. Moreover, the gain of Sensitivity from the minority samples with pseudo-negative samples grows with the improvement of prediction accuracy on all dataset.
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Biologia Computacional/métodos , Sensibilidade e EspecificidadeRESUMO
Lipid raft is an important element for the cellular entry of some viruses, including coronavirus infectious bronchitis virus (IBV). However, the exact role of lipid rafts in the cellular membrane during the entry of IBV into host cells is still unknown. In this study, we biochemically fractionated IBV-infected cells via sucrose density gradient centrifugation after depleting plasma membrane cholesterol with methyl-ß-cyclodextrin or Mevastatin. Our results demonstrated that unlike IBV non-structural proteins, IBV structural proteins co-localized with lipid raft marker caveolin-1. Infectivity assay results of Vero cells illustrated that the drug-induced disruption of lipid rafts significantly suppressed IBV infection. Further studies revealed that lipid rafts were not required for IBV genome replication or virion release at later stages. However, the drug-mediated depletion of lipid rafts in Vero cells before IBV attachment significantly reduced the expression of viral structural proteins, suggesting that drug treatment impaired the attachment of IBV to the cell surface. Our results indicated that lipid rafts serve as attachment factors during the early stages of IBV infection, especially during the attachment stage.
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Vírus da Bronquite Infecciosa/fisiologia , Microdomínios da Membrana/metabolismo , Células A549 , Animais , Caveolina 1/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Colesterol/metabolismo , Endocitose , Humanos , Vírus da Bronquite Infecciosa/genética , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/virologia , Microscopia de Fluorescência , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real , Células Vero , Proteínas do Envelope Viral/metabolismo , Replicação Viral , beta-Ciclodextrinas/farmacologiaRESUMO
A newly emerged pandemic Sydney GII.4-like norovirus (NoV) (Jingzhou GII.4) was isolated in Jingzhou, China in April, 2013, demonstrating the rapid spread of the variant to China. The complete nucleotide sequence was compared with the prototype Sydney 2012 variant and its VP1 gene with that of Huzhou strain (isolated in January 2013 in Huzhou, China). The result demonstrates that the new variant has evolved rapidly, including mutations in the hypervariable P2 domain of the major capsid protein VP1. Our study also shows that the new Jingzhou GII.4 variant co-circulated with GII.3 and GI.2 at the same time, supporting further monitoring of the evolution of the new NoV variant in China.
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Norovirus/genética , China , Genes Virais , Genótipo , Norovirus/classificação , FilogeniaRESUMO
Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is a highly contagious chicken disease, and can lead to serious economic losses in poultry enterprises. The continual introduction of new IBV serotypes requires alternative strategies for the production of timely and safe vaccines against the emergence of variants. Modification of the IBV genome using reverse genetics is one way to generate recombinant IBVs as the candidates of new IBV vaccines. In this study, the recombinant IBV is developed by replacing the ectodomain region of the S1 gene of the IBV Beaudette strain with the corresponding fragment from H120 strain, designated as rBeau-H120(S1e). In Vero cells, the virus proliferates as its parental virus and can cause syncytium formation. The peak titer would reach 10(5.9) 50% (median) tissue culture infective dose/mL at 24 h post-infection. After inoculation of chickens with the recombinant virus, it demonstrated that rBeau-H120(S1e) remained nonpathogenic and was restricted in its replication in vivo. Protection studies showed that vaccination with rBeau-H120 (S1e) at 7-day after hatch provided 80% rate of immune protection against challenge with 10(3) 50% embryos infection dose of the virulent IBV M41 strain. These results indicate that rBeau-H120 (S1e) has the potential to be an alternative vaccine against IBV based on excellent propagation property and immunogenicity. This finding might help in providing further information that replacement of the ectodomain fragment of the IBV Beaudette S1 gene with that from a present field strain is promising for IBV vaccine development.
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Vírus da Bronquite Infecciosa/imunologia , Animais , Galinhas , Infecções por Coronavirus/imunologia , Vírus da Bronquite Infecciosa/genética , Doenças das Aves Domésticas/imunologia , Vacinas Virais/imunologiaRESUMO
Acyl shifts involving N-S and S-S rearrangements are reactions central to the breaking of a peptide bond and forming of thioester intermediates in an intein-catalyzed protein splicing that ultimately leads to the formation of a new peptide bond by an uncatalyzed S-N acyl shift reaction. To mimic these three acyl shift reactions in forming thioesters and the subsequent peptide ligation, here we describe the development of two 9-fluorenylmethoxycarbonyl (Fmoc)-compatible thioester surrogates that can undergo uncatalyzed N-S, S-S, and S-N acyl shifts for preparing thioesters and cyclic peptides. These surrogates were incorporated as a C-terminal amido moiety of a target peptide using Fmoc chemistry by solid-phase synthesis, and then transformed into a thioester or thiolactones via two acyl shift reactions with or without the presence of an external thiol under acidic conditions. The proposed intein-mimetic thioester surrogates were prepared using readily available starting materials including N-methyl cysteine or 2-thioethylbutylamide. A key functional moiety shared in their design is the thioethylamido (TEA) moiety, which is essential to effect a proximity-driven N-S acyl shift under a favorable five-member ring transition in the breaking of a peptide bond. Thus, the tandem series of acyl shifts effected by a TEA moiety in a thioester surrogate together with a thioethylamino moiety of an N-terminal Cys residue in a linear peptide precursor are chemical mimics of an intein, as they mediate both excision and ligation reactions in forming cyclic peptides including cyclic conotoxin and sunflower trypsin inhibitor described herein.
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Biomimética , Peptídeos Cíclicos , Peptídeos/química , Peptídeos Cíclicos/química , Técnicas de Síntese em Fase Sólida , Compostos de Sulfidrila/químicaRESUMO
Foreign nucleic acids, the essential signature molecules of invading pathogens that act as danger signals for host cells, are detected by endosomal nucleic acid-sensing toll-like receptors (TLRs) 3, 7, 8, 9, and 13. These TLRs have evolved to recognize 'non-self' nucleic acids within endosomal compartments and rapidly initiate innate immune responses to ensure host protection through induction of type I interferons, inflammatory cytokines, chemokines, and co-stimulatory molecules and maturation of immune cells. In this review, we highlight our understanding of the recognition of pathogen-associated nucleic acids and activation of corresponding signaling pathways through endosomal nucleic acid-sensing TLRs 3, 7, 8, 9, and 13 for an enormous diversity of pathogens, with particular emphasis on their compartmentalization, intracellular trafficking, proteolytic cleavage, autophagy, and regulatory programs.
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Endossomos/imunologia , Ácidos Nucleicos/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Bactérias/genética , Bactérias/imunologia , Endossomos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Vírus/genética , Vírus/imunologiaRESUMO
Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon-ß (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Bruton's tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-ß upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to d-galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling.
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Antivirais/imunologia , Vírus da Dengue/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptor 3 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Citocinas/biossíntese , Vírus da Dengue/fisiologia , Ativação Enzimática , Proteínas Ativadoras de GTPase/metabolismo , Células HEK293 , Humanos , Interferon beta/biossíntese , Sistema de Sinalização das MAP Quinases , Ativação de Macrófagos , Macrófagos/enzimologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/deficiência , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismo , Replicação ViralRESUMO
Pdcd4 is a tumour suppressor protein. It inhibits translation through interaction with translation initiator eIF4A, resulting in the suppression of neoplastic transformation and tumour invasion. Here, we present the crystal structures of an N-terminal-truncated Pdcd4 in free form and in complex with eIF4A. Upon binding to eIF4A, Pdcd4 undergoes a marked conformational change to form a heterotrimeric complex with eIF4A, with one Pdcd4 binding to two eIF4A molecules in two different modes. The binding of Pdcd4 to eIF4A is required to inhibit the enzymatic activity of eIF4A, translation initiation, and AP-1-dependent transcription. Both MA3 domains are required to efficiently compete with the C-terminal domain of eIF4G (eIF4Gc) for binding to eIF4A whereas a single MA3 is sufficient to inhibit translation. Our structural and mutational analyses reveal that Pdcd4 inhibits translation initiation by trapping eIF4A in an inactive conformation, and blocking its incorporation into the eIF4F complex.
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Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , DNA Helicases/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/química , Fator de Iniciação Eucariótico 4G/metabolismo , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Soluções , Fator de Transcrição AP-1/metabolismo , Transcrição GênicaRESUMO
Plant RNA viruses exploit nonorthodox strategies, such as the use of internal ribosomal entry sites (IRES), to express multiple genes from a single RNA species. IRES elements have been reported in tobacco etch virus (TEV), crucifer infecting tobamovirus (crTMV), hibiscus chlorotic ringspot virus (HCRSV), and many other animal and plant RNA viruses. In this chapter, the methodology used to identify and characterize a plant virus IRES element, including construction of a translation reporter vector for testing the IRES activity, testing the IRES activity in coupled in vitro transcription and translation systems and mammalian cells analysis of RNA stability, and sucrose gradient analysis and polysome profiling, is presented.
Assuntos
Vírus de Plantas/genética , Vírus de RNA/genética , Ribossomos/genética , Animais , Células COS , Chlorocebus aethiops , Técnicas Genéticas , Haplorrinos , Indicadores e Reagentes , Vírus de Plantas/classificação , Biossíntese de Proteínas , Vírus de RNA/classificação , RNA Viral/genética , Ribossomos/metabolismo , Transcrição GênicaRESUMO
Tumour cells of undifferentiated nasopharyngeal carcinoma (NPC) consistently harbour Epstein-Barr virus (EBV) genes. Expression of mRNA transcripts associated with EBV latency has been demonstrated in such cells. However, expression of EBV lytic genes has not been well elucidated, although various lines of evidence have suggested that there is EBV replication in NPC tumour cells. We have studied mRNA expression of representative EBV lytic genes by RT-PCR in nasopharynx biopsies obtained from NPC and control individuals. In both NPC and control biopsies, EBV lytic genes BZLF1, BALF2 and BCLF1 were detected readily. However, BRLF1 was detected in NPC biopsies only. The BRLF1 gene was then cloned and expressed in vitro, and the protein product, Rta, was used as an antigen to detect specific antibodies by immunoprecipitation in plasma samples obtained from NPC patients and healthy controls. IgG antibodies directed against Rta were detected in 44 of 53 NPC plasma samples (83.0%), but only in 1 of 53 control samples (1.9%). Furthermore, the antibody binding regions were found in the C-terminal two-thirds of Rta. This serological result confirms indirectly that BRLF1 is specifically expressed in NPC tumour cells. Rta might play an important role in NPC pathogenesis, considering its multiple functions in EBV replication and cell cycles. Moreover, the detection of IgG antibodies directed against Rta could be developed into a diagnostic parameter for NPC.