RESUMO
Low pathogenic avian influenza (LPAI) viruses are a source of sporadic human infections and could also contribute to future pandemic outbreaks but little is known about inter-species differences in the host responses to these viruses. Here, we studied host gene expression signatures of cell lines from three species (human, chicken, and canine) in response to six different viruses (H1N1/WSN, H5N2/F59, H5N2/F118, H5N2/F189, H5N3 and H9N2). Comprehensive microarray probe set re-annotation and ortholog mapping of the host genes was necessary to allow comparison over extended functionally annotated gene sets and orthologous pathways. The annotations are made available to the community for commonly used microarray chips. We observe a strong tendency of the response being cell type- rather than virus-specific. In chicken cells, we found up-regulation of host factors inducing virus infectivity (e.g., oxysterol binding protein like 1A (OSBPL1A) and Rho GTPase activating protein 21 (ARHGAP21)) while reducing apoptosis (e.g., mitochondrial ribosomal protein S27 (MRPS27)) and increasing cell proliferation (e.g., COP9 signalosome subunit 2 (COPS2)). On the other hand, increased antiviral, pro-apoptotic and inflammatory signatures have been identified in human cells while cell cycle and metabolic pathways were down-regulated. This signature describes how low pathogenic avian influenza (LPAI) viruses are being tolerated and shed from chicken but potentially causing cellular disruption in mammalian cells.
Assuntos
Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/genética , Transcriptoma , Animais , Apoptose , Linhagem Celular , Galinhas , Cães , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H5N2/fisiologia , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/genética , Influenza Aviária/metabolismo , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Redes e Vias Metabólicas , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/virologia , Especificidade da EspécieRESUMO
BACKGROUND: In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens. METHODS: Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted. RESULTS: Electron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range. CONCLUSIONS: The etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.
Assuntos
Vírus da Encefalomiocardite/classificação , Vírus da Encefalomiocardite/isolamento & purificação , Pongo/virologia , Animais , Animais de Zoológico , Chlorocebus aethiops , Análise por Conglomerados , Vírus da Encefalomiocardite/genética , Genoma Viral , Coração/virologia , Pulmão/virologia , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Análise de Sequência de DNA , Homologia de Sequência , Singapura , Células Vero , Proteínas Virais/genética , Vírion/ultraestrutura , Cultura de VírusRESUMO
In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of beta-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.
Assuntos
Proteínas de Choque Térmico HSP90/química , Vírus Sinciciais Respiratórios/fisiologia , Vírion/fisiologia , Montagem de Vírus/fisiologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP90/fisiologia , Imunoprecipitação , RNA Interferente Pequeno , Vírion/químicaRESUMO
In humans, (A549) cells impaired H9N2 virus nuclear export of the ribonucleoprotein (RNP) complex contrasted with the early and efficient nuclear export of the H1N1/WSN and pH1N1 virus RNP complexes. Although nuclear export of the RNP complex occurred via the nuclear pore complex, H9N2 virus infection also induced modifications in the nuclear envelope and induced cell cytotoxicity. Reduced PA protein levels in H9N2 virus-infected A549 cells occurred, and this phenomenon was independent of virus infection. Silencing the H1N1/WSN PA protein expression leads to impaired nuclear export of RNP complexes, suggesting that the impaired nuclear export of the H9N2 virus RNP complex may be one of the consequences of reduced PA protein levels. Early and efficient export of the RNP complex occurred in H9N2 virus-infected avian (CEF) cells, although structural changes in the nuclear envelope also occurred. Collectively our data suggest that a combination of delayed nuclear export and virus-induced cell cytotoxicity restricts H9N2 virus transmission in A549 cells. However, the early and efficient export of the RNP complex mitigated the effects of virus-induced cytotoxicity on H9N2 virus transmission in CEF cells. Our findings highlight the multi-factorial nature of host-adaptation of the polymerase proteins of avian influenza viruses in non-avian cell environments.
Assuntos
Núcleo Celular/metabolismo , Patos/virologia , Vírus da Influenza A Subtipo H9N2/fisiologia , Pulmão/patologia , Pulmão/virologia , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Morte Celular , Linhagem Celular , Galinhas , Humanos , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Although the influenza A virus H7N9 subtype circulates within several avian species, it can also infect humans with a severe disease outcome. To better understand the biology of the H7N9 virus we examined the host response to infection in avian and human cells. In this study we used the A/Anhui/1/2013 strain, which was isolated during the first wave of the H7N9 epidemic. The H7N9 virus-infected both human (Airway Epithelial cells) and avian (Chick Embryo Fibroblast) cells, and each infected host transcriptome was examined with bioinformatic tools and compared with other representative avian and human influenza A virus subtypes. The H7N9 virus induced higher expression changes (differentially regulated genes) in both cell lines, with more prominent changes observed in avian cells. Ortholog mapping of differentially expression genes identified significant enriched common and cell-type pathways during H7N9 infections. This data confirmed our previous findings that different influenza A virus subtypes have virus-specific replication characteristics and anti-virus signaling in human and avian cells. In addition, we reported for the first time, the new HIPPO signaling pathway in avian cells, which we hypothesized to play a vital role to maintain the antiviral state of H7N9 virus-infected avian cells. This could explain the absence of disease symptoms in avian species that tested positive for the presence of H7N9 virus.
Assuntos
Subtipo H7N9 do Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , Células A549 , Animais , Embrião de Galinha , Galinhas , Cães , Expressão Gênica , Humanos , Influenza Aviária/genética , Influenza Aviária/metabolismo , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/metabolismo , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/metabolismo , Transdução de SinaisRESUMO
The study of dynamic movement and interactions of proteins inside living cells in real time is critical for a better understanding of cellular mechanisms and functions in molecular detail. Genetically encoded fusions to fluorescent protein(s) (FP) have been widely used for this purpose [Annu. Rev. Biochem. 1998, 67, 509-544]. To obviate some of the drawbacks associated with the use of FPs [Curr. Opin. Biotechnol. 2005, 16, 1-6; Nat. Methods2006, 3, 591-596], we report a small molecule-based approach that exploits the unique reactivity between the cysteine residue at the N-terminus of a target protein and cell-permeable, thioester-based small molecule probes resulting in site-specific, covalent tagging of proteins. This approach has been demonstrated by the in vivo labeling of proteins in both bacterial and mammalian systems thereby making it potentially useful for future bioimaging applications.
Assuntos
Sondas Moleculares/química , Proteínas Recombinantes de Fusão/análise , Linhagem Celular , Cisteína/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Inteínas , Microscopia de Fluorescência , Sondas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
In the advent of an influenza virus pandemic it is likely that the administration of antiviral drugs will be an important first line of defence against the virus. The drugs currently in use are effective against seasonal influenza virus infection, and some cases have been used in the treatment of patients infected with the avian H5N1 influenza virus. However, it is becoming clear that the emergence of drug-resistant viruses will potentially be a major problem in the future efforts to control influenza virus infection. In addition, during a new pandemic, sufficient quantities of these agents will need to be distributed to many different parts of the world, possibly at short notice. In this review we provide an overview of some of the drugs that are currently available for the treatment and prevention of influenza virus infection. In addition, basic research on influenza virus is providing a much better understanding of the biology of the virus, which is offering the possibility of new anti-influenza virus drugs. We therefore also review some new antiviral strategies that are being reported in the scientific literature, which may form the basis of the next generation of antiviral strategies during a future influenza virus pandemic.
Assuntos
Antivirais/uso terapêutico , Surtos de Doenças/estatística & dados numéricos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/prevenção & controle , Influenza Humana/tratamento farmacológico , Amantadina/uso terapêutico , Animais , Aves , Saúde Global , Humanos , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Oseltamivir/uso terapêutico , RNA Interferente PequenoRESUMO
We review various advancements in small molecule probes, intein-based labeling methods, and the incorporation of synthetic amino acids into proteins for live cell imaging experiments. Finally, recent developments in quantum dots-based labeling are briefly reviewed.
Assuntos
Células/citologia , Diagnóstico por Imagem , Animais , Técnicas Citológicas , Corantes Fluorescentes , Humanos , Sondas Moleculares/química , NanotecnologiaRESUMO
Recent advances in the generation of peptide and protein microarrays are reviewed, with special focuses on different strategies available for site-specific immobilization of proteins and peptides.
Assuntos
Análise Serial de Proteínas/métodos , Avidina , Biotinilação , DNA Polimerase Dirigida por DNA/química , Peptídeos/química , Proteínas/química , Proteômica , PuromicinaRESUMO
We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells. Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo.
Assuntos
Permeabilidade da Membrana Celular , Sondas Moleculares/química , Proteínas/química , Proteínas/metabolismo , Cisteína/química , Escherichia coli/química , Corantes Fluorescentes/química , Regulação da Expressão Gênica , Sondas Moleculares/síntese química , Estrutura MolecularRESUMO
The host response to the low pathogenic avian influenza (LPAI) H5N2, H5N3 and H9N2 viruses were examined in A549, MDCK, and CEF cells using a systems-based approach. The H5N2 and H5N3 viruses replicated efficiently in A549 and MDCK cells, while the H9N2 virus replicated least efficiently in these cell types. However, all LPAI viruses exhibited similar and higher replication efficiencies in CEF cells. A comparison of the host responses of these viruses and the H1N1/WSN virus and low passage pH1N1 clinical isolates was performed in A549 cells. The H9N2 and H5N2 virus subtypes exhibited a robust induction of Type I and Type III interferon (IFN) expression, sustained STAT1 activation from between 3 and 6 hpi, which correlated with large increases in IFN-stimulated gene (ISG) expression by 10 hpi. In contrast, cells infected with the pH1N1 or H1N1/WSN virus showed only small increases in Type III IFN signalling, low levels of ISG expression, and down-regulated expression of the IFN type I receptor. JNK activation and increased expression of the pro-apoptotic XAF1 protein was observed in A549 cells infected with all viruses except the H1N1/WSN virus, while MAPK p38 activation was only observed in cells infected with the pH1N1 and the H5 virus subtypes. No IFN expression and low ISG expression levels were generally observed in CEF cells infected with either AIV, while increased IFN and ISG expression was observed in response to the H1N1/WSN infection. These data suggest differences in the replication characteristics and antivirus signalling responses both among the different LPAI viruses, and between these viruses and the H1N1 viruses examined. These virus-specific differences in host cell signalling highlight the importance of examining the host response to avian influenza viruses that have not been extensively adapted to mammalian tissue culture.
Assuntos
Células Epiteliais/metabolismo , Influenza Humana/patologia , Interferon Tipo I/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Reguladoras de Apoptose , Aves , Linhagem Celular Tumoral , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H5N2/genética , Vírus da Influenza A Subtipo H5N2/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/crescimento & desenvolvimento , Influenza Aviária/genética , Influenza Aviária/virologia , Influenza Humana/enzimologia , Interferon Tipo I/genética , Interferons , Interleucinas/genética , Interleucinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Viral/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT1/metabolismo , Replicação Viral , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We examined the structure of lipid-raft membranes in respiratory syncytial virus infected cells. Cholesterol depletion studies using methyl-beta-cyclodextrin suggested that membrane cholesterol was required for virus filament formation, but not inclusion bodies. In addition, virus filament formation coincided with elevated 3-hydroxy-3-methylglutaryl-coenzyme A reductase expression, suggesting an increase in requirement for endogenous cholesterol synthesis during virus assembly. Lipid raft membranes were examined by mass spectrometry, which suggested that virus infection induced subtle changes in the lipid composition of these membrane structures. This analysis revealed increased levels of raft-associated phosphatidylinositol (PI) and phosphorylated PI during RSV infection, which correlated with the appearance of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate (PIP(3)) within virus inclusion bodies, and inhibiting the synthesis of PIP(3) impaired the formation of progeny virus. Collectively, our analysis suggests that RSV infection induces specific changes in the composition of raft-associated lipids, and that these changes play an important role in virus maturation.
Assuntos
Metabolismo dos Lipídeos , Lipídeos/análise , Microdomínios da Membrana/química , Vírus Sinciciais Respiratórios/fisiologia , Montagem de Vírus , Linhagem Celular , Humanos , Hidroximetilglutaril-CoA-Redutases NADP-Dependentes/metabolismo , Corpos de Inclusão Viral/química , Espectrometria de MassasRESUMO
We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.
Assuntos
Comércio , Microbiologia de Alimentos , Vírus da Influenza A/isolamento & purificação , Influenza Aviária/virologia , Animais , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Influenza Aviária/epidemiologia , Modelos Moleculares , Neuraminidase/genética , Neuraminidase/metabolismo , Filogenia , Vigilância da População , Aves Domésticas , Conformação Proteica , Singapura/epidemiologiaRESUMO
One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. Herein, we report a bacterium-based, intein-mediated strategy to generate N-terminal cysteine-containing proteins which are then chemoselectively immobilized to a thioester-functionalized glass slide to generate the corresponding protein microarray. We also showed preliminary data of the strategy in a yeast host system.
Assuntos
Inteínas , Análise Serial de Proteínas , Proteínas/química , Eletroforese em Gel de PoliacrilamidaRESUMO
The post-genomic era heralds a multitude of challenges for chemists and biologists alike, with the study of protein functions at the heart of much research. The elucidation of protein structure, localization, stability, post-translational modifications, and protein interactions will steadily unveil the role of each protein and its associated biological function in the cell. The push to develop new technologies has necessitated the integration of various disciplines in science. Consequently, the role of chemistry has never been so profound in the study of biological processes. By combining the strengths of recombinant DNA technology, protein splicing, organic chemistry, and the chemoselective chemistry of native chemical ligation, various strategies have been successfully developed and applied to chemoselectively label proteins, both in vitro and in live cells, with biotin, fluorescent, and other small molecule probes. The site-specific incorporation of molecular entities with unique chemical functionalities in proteins has many potential applications in chemical and biological studies of proteins. In this article, we highlight recent progress of these strategies in several areas related to proteomics and chemical biology, namely, in vitro and in vivo protein biotinylation, protein microarray technologies for large-scale protein analysis, and live-cell bioimaging.