RESUMO
BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ciclo Celular , Proteínas de Membrana Transportadoras , Viroses , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/genética , Autofagia/genética , Proteínas de Ciclo Celular/genética , Imunidade Inata , Interferon beta/genética , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos KnockoutRESUMO
One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays reveal that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of approximately 17,000 SARS-CoV-2 sequences identify a natural variant in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients but also describe the emergence of natural SARS-CoV-2 quasispecies with an extended ORF3b gene that may potentially affect COVID-19 pathogenesis.
Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Interferon Tipo I/antagonistas & inibidores , Pneumonia Viral/virologia , Proteínas Virais Reguladoras e Acessórias/genética , Adulto , Sequência de Aminoácidos/genética , Animais , Betacoronavirus/imunologia , COVID-19 , Quirópteros/virologia , Códon sem Sentido/genética , Infecções por Coronavirus/patologia , Eutérios/virologia , Humanos , Masculino , Pandemias , SARS-CoV-2 , Proteínas Virais Reguladoras e Acessórias/metabolismoRESUMO
The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.
Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Imunidade Inata/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Psoríase/prevenção & controle , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Células A549 , Animais , Anti-Inflamatórios/química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Feminino , Células HEK293 , Células HeLa , Humanos , Imiquimode , Mediadores da Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Psoríase/induzido quimicamente , Psoríase/imunologia , Psoríase/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
An influenza virus epidemic is an important issue in public hygiene, and continuous development on an effective drug is required. Kampo medicine is a traditional medicine that is used clinically for treatment of various diseases in Japan and other East Asian countries. We evaluated the effects of the Kampo drugs maoto, kakkonto, senkyuchachosan, jinkokato, and bakumondoto, which are prescribed for treatment of respiratory symptoms including symptoms caused by influenza, on influenza virus replication in cultured cells. Culture media of influenza virus-infected MDCK(+) cells were tested for hemagglutination and infectivity at 24 h after the addition of Kampo drugs at various concentrations, and four of the five Kampo drugs were found to inhibit virus release to the culture media. These drugs inactivated virus infectivity not by acting on virus particles but by acting on virus-infected cells. In addition, when six crude drugs (Atractylodis lanceae rhizome, Citri unshiu pericarpium, Cnidii rhizome, Glycyrrhizae radix, Rehmanniae radix, and Saposhnikoviae radix) that constitute the effective Kampo drugs were examined, the strongest activity was found for Glycyrrhizae radix (IC50 = 0.27 mg/ml), which selectively suppressed viral protein synthesis. Since Glycyrrhizae radix is contained in many Kampo drugs, it may give anti-influenza virus activity to a broad range of Kampo drugs.
RESUMO
Feline calicivirus (FCV) is frequently used as a surrogate of human norovirus. We investigated eligibility of FCV for anti-viral assay by investigating the stability of infectivity and pH sensitivity in comparison with other viruses. We found that infectivities of FCV and murine norovirus (MNV) are relatively unstable in infected cells compared with those of coxsackievirus (CoV) and poliovirus (PoV) , suggesting that FCV and MNV have vulnerability. Western blotting indicated that inactivation of FCV was not due to viral protein degradation. We also demonstrated sensitivity of FCV to low pH, the 50% inhibitory pH value being ca. 3.9. Since human norovirus is thought to persist longer, in infectivity and to be a resistant virus, CoV, which is robust and not restrained in use as PoV, may be more appropriate as a test virus for disinfectants, rather than FCV and MNV.
Assuntos
Calicivirus Felino/fisiologia , Enterovirus/fisiologia , Células Epiteliais/virologia , Norovirus/fisiologia , Poliovirus/fisiologia , Carga Viral , Animais , Calicivirus Felino/patogenicidade , Gatos , Linhagem Celular , Enterovirus/patogenicidade , Células Epiteliais/patologia , Humanos , Concentração de Íons de Hidrogênio , Rim/patologia , Rim/virologia , Camundongos , Modelos Biológicos , Norovirus/patogenicidade , Células-Tronco Pluripotentes/patologia , Células-Tronco Pluripotentes/virologia , Poliovirus/patogenicidade , Células RAW 264.7 , Replicação ViralRESUMO
UNLABELLED: Sendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/ß) and IFN-γ by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr(701) by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the γ-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-γ by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-α/ß. IMPORTANCE: Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/ß) and IFN-γ by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr(701) residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr(701), further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-γ. We also discuss the possible mechanism of inhibition of the IFN-α/ß pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.
Assuntos
Interferon-alfa/antagonistas & inibidores , Interferon beta/antagonistas & inibidores , Interferon gama/antagonistas & inibidores , Fator de Transcrição STAT1/antagonistas & inibidores , Proteínas Virais/ultraestrutura , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Humanos , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/ultraestrutura , Fator de Transcrição STAT2/metabolismo , Vírus Sendai/metabolismo , Transdução de Sinais/fisiologia , Proteínas Virais/metabolismoRESUMO
Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, a fatal pulmonary disorder with no effective treatment. We found that SARS-CoV spike glycoprotein (S protein), a key molecule for viral entry, binds to calnexin, a molecular chaperone in the endoplasmic reticulum (ER), but not to calreticulin, a homolog of calnexin. Calnexin bound to most truncated mutants of S protein, and S protein bound to all mutants of calnexin. Pseudotyped virus carrying S protein (S-pseudovirus) produced by human cells that were treated with small interfering RNA (siRNA) for calnexin expression (calnexin siRNA-treated cells) showed significantly lower infectivity than S-pseudoviruses produced by untreated and control siRNA-treated cells. S-pseudovirus produced by calnexin siRNA-treated cells contained S protein modified with N-glycan side chains differently from other two S proteins and consisted of two kinds of viral particles: those of normal density with little S protein and those of high density with abundant S protein. Treatment with peptide-N-glycosidase F (PNGase F), which removes all types of N-glycan side chains from glycoproteins, eliminated the infectivity of S-pseudovirus. S-pseudovirus and SARS-CoV produced in the presence of α-glucosidase inhibitors, which disrupt the interaction between calnexin and its substrates, showed significantly lower infectivity than each virus produced in the absence of those compounds. In S-pseudovirus, the incorporation of S protein into viral particles was obviously inhibited. In SARS-CoV, viral production was obviously inhibited. These findings demonstrated that calnexin strictly monitors the maturation of S protein by its direct binding, resulting in conferring infectivity on SARS-CoV.
Assuntos
Calnexina/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Animais , Linhagem Celular , Glicosilação , Humanos , Camundongos , Ligação Proteica , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Glicoproteína da Espícula de CoronavírusRESUMO
BACKGROUND: Patients with influenza virus infection can develop severe pneumonia and acute respiratory distress syndrome (ARDS) which have a high mortality. Influenza virus infection is treated worldwide mainly by neuraminidase inhibitors (NAIs). However, monotherapy with NAIs is insufficient for severe pneumonia secondary to influenza virus infection. We previously demonstrated that mice infected with a lethal dose of influenza virus develop diffuse alveolar damage (DAD) with alveolar collapse similar to that seen in ARDS in humans. Additionally, pulmonary surfactant proteins were gradually increased in mouse serum, suggesting a decrease in pulmonary surfactant in the lung. Therefore, the present study examined whether combination therapy of NAI with exogenous artificial surfactant affects mortality of influenza virus-infected mice. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c mice were inoculated with several viral doses of influenza A/Puerto Rico/8/34 (PR8) virus (H1N1). The mice were additionally administered exogenous artificial surfactant in the presence or absence of a new NAI, laninamivir octanoate. Mouse survival, body weight and general condition were observed for up to 20 days after inoculation. Viral titer and cytokine/chemokine levels in the lungs, lung weight, pathological analysis, and blood O(2) and CO(2) pressures were evaluated. Infected mice treated with combination therapy of laninamivir octanoate with artificial surfactant showed a significantly higher survival rate compared with those that received laninamivir octanoate monotherapy (p = 0.003). However, virus titer, lung weight and cytokine/chemokine responses were not different between the groups. Histopathological examination, a hydrostatic lung test and blood gas analysis showed positive results in the combination therapy group. CONCLUSIONS/SIGNIFICANCE: Combination therapy of laninamivir octanoate with artificial surfactant reduces lethality in mice infected with influenza virus, and eventually suppresses DAD formation and preserves lung function. This combination could be effective for prevention of severe pneumonia secondary to influenza virus infection in humans, which is not improved by NAI monotherapy.
Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Surfactantes Pulmonares/uso terapêutico , Zanamivir/análogos & derivados , Animais , Peso Corporal/efeitos dos fármacos , Dióxido de Carbono/sangue , Quimiocinas/metabolismo , Cães , Quimioterapia Combinada , Feminino , Guanidinas , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Tamanho do Órgão/efeitos dos fármacos , Infecções por Orthomyxoviridae/imunologia , Oxigênio/sangue , Pressão , Proteína D Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/administração & dosagem , Piranos , Ácidos Siálicos , Análise de Sobrevida , Carga Viral/efeitos dos fármacos , Zanamivir/farmacologia , Zanamivir/uso terapêuticoRESUMO
Myelination is essential for proper functioning of the CNS. In this study, we have identified a mouse mutation, designated furue, which causes tremors and hypomyelination in the CNS, particularly in the spinal cord, but not in the sciatic nerve of the PNS. In the spinal cord of the furue mice, myelination of small-diameter axons was dramatically reduced, and differentiation of oligodendrocytes, the myelin-forming cells in the CNS, was inhibited. We subsequently found that the furue mutation was associated with a transgene insertion into the teneurin-4 (Ten-4, Ten-m4/Odz4) gene, encoding a transmembrane protein of unknown function. Ten-4 was strongly expressed in the spinal cord of wild-type mice and was induced during normal oligodendrocyte differentiation. In contrast, in the furue mice, the expression of Ten-4 was absent. Differentiation and cellular process formation of oligodendrocytes were inhibited in primary cell culture from the furue mice. Cell differentiation and process formation were also inhibited in the oligodendrocyte progenitor cell line CG-4 after suppression of Ten-4 expression by shRNA. Furthermore, Ten-4 positively regulated focal adhesion kinase, an essential signaling molecule for oligodendrocyte process formation and myelination of small-diameter axons. These findings suggest that Ten-4 is a novel regulator of oligodendrocyte differentiation and that it plays a critical role in the myelination of small-diameter axons in the CNS.
Assuntos
Axônios/metabolismo , Diferenciação Celular/genética , Sistema Nervoso Central , Doenças Desmielinizantes/genética , Proteínas Nucleares/deficiência , Oligodendroglia/citologia , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Antígenos/metabolismo , Axônios/patologia , Axônios/ultraestrutura , Encéfalo/citologia , Tamanho Celular , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Galactosilceramidase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteína Básica da Mielina/metabolismo , Neuroglia/fisiologia , Proteínas Nucleares/genética , Organogênese , Proteoglicanas/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , TransfecçãoRESUMO
Avian influenza H5N1 and pandemic (H1N1) 2009 viruses are known to induce viral pneumonia and subsequent acute respiratory distress syndrome (ARDS) with diffuse alveolar damage (DAD). The mortality rate of ARDS/DAD is extremely high, at approximately 60%, and no effective treatment for ARDS/DAD has been established. We examined serial pathological changes in the lungs of mice infected with influenza virus to determine the progress from viral pneumonia to ARDS/DAD. Mice were intranasally infected with influenza A/Puerto Rico/8/34 (PR8) virus, and their lungs were examined both macro- and micro-pathologically every 2 days. We also evaluated general condition, survival rate, body weight, viral loads in lung, and surfactant proteins in serum. As a result, all infected mice died within 9 days postinfection. At 2 days postinfection, inflammation in alveolar septa, i.e., interstitial pneumonia, was observed around bronchioles. From 4 to 6 days postinfection, interstitial pneumonia with alveolar collapse expanded throughout the lungs. From 6 to 9 days postinfection, DAD with severe alveolar collapse was observed in the lungs of all of dying and dead mice. In contrast, DAD was not observed in the live infected-mice from 2 to 6 days postinfection, despite their poor general condition. In addition, histopathological analysis was performed in mice infected with a dose of PR8 virus which was 50% of the lethal dose for mice in the 20-day observation period. DAD with alveolar collapse was observed in all dead mice. However, in the surviving mice, instead of DAD, glandular metaplasia was broadly observed in their lungs. The present study indicates that DAD with severe alveolar collapse is associated with death in this mouse infection model of influenza virus. Inhibition of the development of DAD with alveolar collapse may decrease the mortality rate in severe viral pneumonia caused by influenza virus infection.
Assuntos
Vírus da Influenza A/patogenicidade , Pulmão/patologia , Pulmão/virologia , Infecções por Orthomyxoviridae/patologia , Animais , Linhagem Celular , Cães , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/virologiaRESUMO
BACKGROUND: There is still no effective method to prevent or treat severe acute respiratory syndrome (SARS), which is caused by SARS coronavirus (CoV). In the present study, we evaluated the efficacy of a fully human monoclonal antibody capable of neutralizing SARS-CoV in vitro in a Rhesus macaque model of SARS. METHODS: The antibody 5H10 was obtained by vaccination of KM mice bearing human immunoglobulin genes with Escherichia coli-producing recombinant peptide containing the dominant epitope of the viral spike protein found in convalescent serum samples from patients with SARS. RESULTS: 5H10, which recognized the same epitope that is also a cleavage site critical for the entry of SARS-CoV into host cells, inhibited propagation of the virus and pathological changes found in Rhesus macaques infected with the virus through the nasal route. In addition, we analyzed the mode of action of 5H10, and the results suggested that 5H10 inhibited fusion between the virus envelope and host cell membrane. 5H10 has potential for use in prevention and treatment of SARS if it reemerges. CONCLUSIONS: This study represents a platform to produce fully human antibodies against emerging infectious diseases in a timely and safe manner.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Membrana/imunologia , Síndrome Respiratória Aguda Grave/terapia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Enzima de Conversão de Angiotensina 2 , Animais , Animais Geneticamente Modificados , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Western Blotting , Domínio Catalítico , Fusão Celular , Modelos Animais de Doenças , Células Gigantes/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Pulmão/patologia , Pulmão/virologia , Macaca mulatta , Glicoproteínas de Membrana/genética , Camundongos , Peptidil Dipeptidase A , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral/genéticaRESUMO
When expressed in mammalian cells, the nucleocapsid (N) and membrane (M) proteins of the severe acute respiratory syndrome coronavirus (SARS-CoV) are sufficient to form pseudoparticles. To identify region(s) of the N molecule required for pseudoparticle formation, we performed biochemical analysis of the interaction of N mutants and M in HEK293 cells. Using a peptide library derived from N, we found that amino acids 101-115 constituted a novel binding site for M. We examined the ability of N mutants to interact with M and form pseudoparticles, and our observations indicated that M bound to NDelta(101-115), N1-150, N151-300, and N301-422, but not to N1-150Delta(101-115). However, pseudoparticles were formed when NDelta(101-115) or N301-422, but not N1-150 or N151-300, were expressed with M in HEK293 cells. These results indicated that the minimum portion of N required for the interaction with M and pseudoparticle formation consists of amino acids 301-422.
Assuntos
Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Proteínas da Matriz Viral/metabolismo , Linhagem Celular , Proteínas M de Coronavírus , Proteínas do Nucleocapsídeo de Coronavírus , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , RNA Viral/química , Proteínas da Matriz Viral/genéticaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) establishes latent infection in various cells in vitro as well as KSHV-associated tumor cells in vivo. The latency-associated nuclear antigen (LANA) of KSHV is one of a small number of genes expressed in the latent phase of KSHV infection. This antigen is crucial for establishment of the latent infection, such as replication of KSHV genomic DNA and maintenance of infection via direct interaction with terminal repeats (TRs) in the viral genome. Using a yeast two-hybrid screening method, we isolated a novel LANA-interacting protein (designated as KZLP; KRAB Zinc finger LANA interacting Protein) from a human peripheral leukocyte cDNA library. KZLP encodes a KRAB domain and 12 Kruppel-type zinc fingers. Reverse transcription polymerase chain reaction showed that KZLP was expressed ubiquitously in various cell lines including those infected with KSHV. A luciferase assay showed that KZLP could activate the KSHV open reading frame K1 promoter containing TRs in 293T cells, and that such activation required multiple TR sequences. In contrast, LANA repressed the activity of the K1 promoter through TRs, and again this repression required multiple TR units. Moreover, LANA almost completely abrogated the KZLP-mediated transcriptional activation. Our results suggest that KZLP and LANA regulate gene expression through TRs in the KSHV viral genome, including the K1 gene in latent KSHV-infected cells.
Assuntos
Antígenos Virais/metabolismo , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Antígenos Virais/genética , Linhagem Celular , Regulação Viral da Expressão Gênica , Células HeLa , Herpesvirus Humano 8/genética , Humanos , Proteínas Nucleares/genética , Sarcoma de Kaposi/fisiopatologia , Sequências Repetidas Terminais , Fatores de Transcrição/metabolismo , Transcrição Gênica , Latência Viral , Dedos de Zinco/fisiologiaRESUMO
The latency-associated nuclear antigen (LANA) of Kaposi's saroma-associated herpesvirus (KSHV) can maintain a plasmid containing the KSHV origin of DNA replication (oriP) as episomes in dividing human cells. Hence, LANA is considered to play crucial roles in persistent KSHV infection in human cells. In this study, we characterized a LANA fusion protein of green fluorescent protein (GFP-LANA). Like the wild-type LANA, GFP-LANA interacted tightly with mitotic chromosomes, and maintained the plasmid selectively with the KSHV oriP for more than three weeks in a human B cell line. Moreover, equivalent amount of GFP-LANA protein was segregated into two daughter cells in living metaphase cells. Our results suggested that the activity of LANA serves the segregation of equivalent amounts of viral genomes tethered with LANA into two daughter progeny cells during cell division. Thus, GFP-LANA is a useful tool for the analyses of the functions and dynamics of LANA in living cells.
Assuntos
Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/metabolismo , Plasmídeos/genética , Origem de Replicação/genética , Antígenos Virais , Linfócitos B , Linhagem Celular , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , Replicação do DNA , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Metáfase , Mitose , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), whereas the closely related virus HTLV-2 has not been associated with such malignant conditions. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) much more efficiently than does HTLV-2 Tax2. By using a differential display analysis, we isolated MAGI-3 as a Tax1-inducible gene in Rat-1 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that Tax1 induced MAGI-3 in Rat-1 cells. MAGI-3 has multiple PDZ domains and interacted with Tax1 but not Tax2 in 293T cells. The interaction of Tax1 with MAGI-3 was dependent on a PDZ domain-binding motif, which is missing in Tax2. The interaction of Tax1 with MAGI-3 altered their respective subcellular localization, and moreover, the interaction correlated well with the high transforming activities of Tax1 in Rat-1 cells relative to Tax2. MAGI-3 mRNA and the allied MAGI-1, but not MAGI-2, were expressed in HTLV-1-infected T-cell lines. Our results suggest that the interaction of Tax1 and MAGI-3 alters their respective biological activities, which may play a role in transformation by Tax1 as well as in the pathogenesis of HTLV-1-associated diseases.
Assuntos
Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Núcleosídeo-Fosfato Quinase/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Transformação Celular Viral , Expressão Gênica , Produtos do Gene tax/análise , Produtos do Gene tax/farmacologia , Guanilato Quinases , Infecções por HTLV-I/etiologia , Vírus Linfotrópico T Tipo 1 Humano/química , Humanos , Espaço Intracelular/metabolismo , Núcleosídeo-Fosfato Quinase/análise , Núcleosídeo-Fosfato Quinase/biossíntese , Núcleosídeo-Fosfato Quinase/genética , Estrutura Terciária de Proteína , RNA Mensageiro/análise , RatosRESUMO
While human T-cell leukemia virus type 1 (HTLV-1) is associated with the development of adult T-cell leukemia (ATL), HTLV-2 has not been reported to be associated with such malignant leukemias. HTLV-1 Tax1 oncoprotein transforms a rat fibroblast cell line (Rat-1) to form multiple large colonies in soft agar, and this activity is much greater than that of HTLV-2 Tax2. We have demonstrated here that the increased number of transformed colonies induced by Tax1 relative to Tax2 was mediated by a PDZ domain-binding motif (PBM) in Tax1, which is absent in Tax2. Tax1 PBM mediated the interaction of Tax1 with the discs large (Dlg) tumor suppressor containing PDZ domains, and the interaction correlated well with the transforming activities of Tax1 and the mutants. Through this interaction, Tax1 altered the subcellular localization of Dlg from the detergent-soluble to the detergent-insoluble fraction in a fibroblast cell line as well as in HTLV-1-infected T-cell lines. These results suggest that the interaction of Tax1 with PDZ domain protein(s) is critically involved in the transforming activity of Tax1, the activity of which may be a crucial factor in malignant transformation of HTLV-1-infected cells in vivo.
Assuntos
Transformação Celular Viral , Fibroblastos/virologia , Produtos do Gene tax/química , Produtos do Gene tax/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas do Citoesqueleto , Proteína 1 Homóloga a Discs-Large , Humanos , Proteínas de Membrana , Dados de Sequência Molecular , Proteínas/metabolismo , Ratos , Frações Subcelulares/metabolismo , Linfócitos T/virologiaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpes virus type 8 (HHV-8) is tightly linked to the development of Kaposi's sarcoma, primary effusion lymphoma (PEL) and some cases of multicentric Castleman's disease. Latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in these diseases as well as in KSHV-infected cell lines derived from PEL, and has been shown to play crucial role in persistence of KSHV genomes in the infected cells. In this study, we explored the cellular factors that interact with LANA using yeast two-hybrid screening, and isolated a part of gene encoding human myeloid cell nuclear differentiation antigen (MNDA). MNDA is a hematopoietic interferon-inducible nuclear proteins with a HIN-200 family member with conserved 200-amino acid repeats. Immunoprecipitation assay revealed that LANA interacted with MNDA in a mammalian embryonic kidney cell line. MNDA transcript was undetectable in three PEL cell lines by reverse-transcription polymerase chain reaction, but it was induced by interferon alpha (IFNalpha). Moreover, LANA and MNDA were co-localized in the nuclei of MNDA-expressing PEL cells. Our results suggest that LANA interacts with MNDA in KSHV-infected cells exposed to IFNalpha. Such interaction may modulate IFN-mediated host defense activities.
Assuntos
Antígenos de Diferenciação Mielomonocítica/biossíntese , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos Virais/genética , Antígenos Virais/fisiologia , Sequência de Bases , Divisão Celular , Linhagem Celular , Núcleo Celular/imunologia , Núcleo Celular/virologia , DNA Viral/genética , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Humanos , Interferon Tipo I/farmacologia , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Recombinantes , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/virologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cell-cell adhesion is involved in the processes of cell growth, activation and migration, and inflammation. T cells infected with human T cell leukemia virus type 1 (HTLV-1) exhibit a high degree of homotypic cell-cell adhesion in vitro. In this study, we investigated the involvement of the viral protein Tax in such process. Expression of Tax in an interleukin (IL)-2-dependent mouse T cell line (CTLL-2) increased homotypic cell-cell adhesion; however, less cell adhesion was induced by Tax than that observed in HTLV-1-infected T cell lines. Moreover, Tax induced cell-cell adhesion in a human T cell line, in which the expression of Tax is inducible. Microscopic examination also revealed Tax-induced morphologic changes, including rounding of CTLL-2 cells, increased cell volume, and increased nucleus size. Taken together, our results suggest that Tax induces cell-cell adhesion and morphologic changes in HTLV-1-infected cells. Tax may thus play a role in persistent HTLV-1 infection and the pathogenesis of associated disease.
Assuntos
Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Animais , Adesão Celular , Linhagem Celular , Produtos do Gene tax/genética , Humanos , Células Jurkat , Camundongos , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (DeltaN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of DeltaN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.
Assuntos
Cromossomos Humanos/metabolismo , Herpesvirus Humano 8/genética , Histonas/fisiologia , Proteínas Nucleares/metabolismo , Plasmídeos , Antígenos Virais , Sítios de Ligação , Proteínas de Transporte/fisiologia , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Humanos , Mitose , Proteínas Nucleares/química , Proteínas Nucleares/fisiologia , Proteínas de Ligação a RNA , Células Tumorais CultivadasRESUMO
Extracellular signal regulated kinase1/2 (ERK1/2), an important factor in signal transduction, controls cell growth, differentiation, and death. To elucidate the details of the mechanism of ERK1/2 signaling in human cells, we isolated Nef-associated factor 1 alpha (Naf1 alpha) by a yeast two-hybrid system, which bound to human ERK2. The binding was confirmed by a pull-down assay in vitro and immunoprecipitation in vivo. Upon EGF treatment, Naf1 alpha was phosphorylated by the EGF/MEK/ERK2 signal transduction pathway. To identify the role of Naf1 alpha in the ERK2 signaling, Naf1 alpha-expressing Saos-2 cells were analyzed for ERK2 nuclear translocation and activation of its downstream target. Overexpression of Naf1 alpha suppressed ERK2 entering into the nucleus and inhibited the ERK2-dependent Elk1-driven luciferase transcription, suggesting Naf1 alpha to be an attenuator of activated ERK2 signaling.