RESUMO
Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.
Assuntos
Quinase I-kappa B/imunologia , Interferon Tipo I/imunologia , Poliubiquitina/biossíntese , Ubiquitina-Proteína Ligases/imunologia , Animais , Antivirais , Células Cultivadas , Ativação Enzimática/imunologia , Humanos , Janus Quinase 1 , Camundongos , Fosforilação/imunologia , Interferência de RNA , RNA Interferente Pequeno , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido , Enzimas de Conjugação de Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Live-attenuated influenza vaccines (LAIVs) have the potential to generate CD8 T cell immunity that may limit the virulence of an antigenically shifted influenza strain in a population lacking protective Abs. However, current LAIVs exert limited T cell immunity restricted to the vaccine strains. One approach to improve LAIV-induced T cell responses is the use of specific adjuvants to enhance T cell priming by respiratory dendritic cells, but this hypothesis has not been addressed. In this study, we assessed the effect of the TLR3 ligand polyinosinic-polycytidylic acid (poly IC) on CD8 T cell immunity and protection elicited by LAIVs. Mucosal treatment with poly IC shortly after vaccination enhanced respiratory dendritic cell function, CD8 T cell formation, and production of neutralizing Abs. This adjuvant effect of poly IC was dependent on amplification of TLR3 signaling by nonhematopoietic radioresistant cells and enhanced mouse protection to homosubtypic, as well as heterosubtypic, virus challenge. Our findings indicate that mucosal TLR3 ligation may be used to improve CD8 T cell responses to replicating vaccines, which has implications for protection in the absence of pre-existing Ab immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Mucosa Nasal/imunologia , Poli I-C/administração & dosagem , Poli I-C/uso terapêutico , Replicação Viral/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/virologia , Células HEK293 , Humanos , Imunidade Celular/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/uso terapêutico , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Poli I-C/imunologia , Regulação para Cima/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
We report that swine influenza virus-like substitutions T200A and E227A in the hemagglutinin (HA) of the 2009 pandemic influenza virus alter its pathogenesis and transmission. Viral replication is increased in mammalian cells. Infected mice show increased disease as measured by weight loss and lethality. Transmission in ferrets is decreased in the presence of both substitutions, suggesting that amino acids 200T and 227E are adaptive changes in the HA of swine origin influenza viruses associated with increased transmission and decreased pathogenesis.
Assuntos
Substituição de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/mortalidade , Influenza Humana/transmissão , Animais , Regulação para Baixo , Feminino , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/epidemiologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos DBA , Pandemias , Regulação para Cima , VirulênciaRESUMO
Influenza A viruses encoding an altered viral NS1 protein have emerged as promising live attenuated vaccine platforms. A carboxy-terminal truncation in the NS1 protein compromises its interferon antagonism activity, making these viruses attenuated in the host yet still able to induce protection from challenge with wild-type viruses. However, specific viral protein expression by NS1-truncated viruses is known to be decreased in infected cells. In this report, we show that recombinant H5N1 and H1N1 influenza viruses encoding a truncated NS1 protein expressed lower levels of hemagglutinin (HA) protein in infected cells than did wild-type viruses. This reduction in HA protein expression correlated with a reduction in HA mRNA levels in infected cells. NS1 truncation affected the expression of HA protein but not that of the nucleoprotein (NP). This segment specificity was mapped to the terminal sequences of their specific viral RNAs. Since the HA protein is the major immunogenic component in influenza virus vaccines, we sought to restore its expression levels in NS1-truncated viruses in order to improve their vaccine efficacy. For this purpose, we generated an NS1-truncated recombinant influenza A/Puerto Rico/8/34 (rPR8) virus carrying the G3A C8U "superpromoter" mutations in the HA genomic RNA segment. This strategy retained the attenuation properties of the recombinant virus but enhanced the expression level of HA protein in infected cells. Finally, mice immunized with rPR8 viruses encoding a truncated NS1 protein and carrying the G3A C8U mutations in the HA segment demonstrated enhanced protection from wild-type virus challenge over that for mice vaccinated with an rPR8 virus encoding the truncated NS1 protein alone.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/virologia , Regulação para Cima , Proteínas não Estruturais Virais/genética , Motivos de Aminoácidos , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Galinhas , Feminino , Regulação Viral da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/química , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/química , Virus da Influenza A Subtipo H5N1/imunologia , Influenza Humana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas não Estruturais Virais/imunologiaRESUMO
Influenza A viruses containing the promoter mutations G3A/C8U in a given segment express increased levels of the corresponding viral protein during infection due to increased levels of mRNA or cRNA species. The replication of these recombinant viruses is attenuated, and they have an enhanced shedding of noninfectious particles and are incapable of antagonizing interferon (IFN) effectively. Our findings highlight the possibility of increasing influenza virus protein expression and the need for a delicate balance between influenza viral replication, protein expression, and assembly.
Assuntos
Regulação Viral da Expressão Gênica , Vírus da Influenza A/enzimologia , Vírus da Influenza A/genética , Influenza Humana/virologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Humanos , Vírus da Influenza A/fisiologia , Mutação , Regiões Promotoras Genéticas , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Replicação ViralRESUMO
Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b(+)CD11c(-), CD11b(-)CD11c(+), and CD11b(+)CD11c(+), as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type-specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field.
Assuntos
Genes Reporter , Proteínas de Fluorescência Verde/genética , Influenza Humana/genética , Infecções por Orthomyxoviridae/metabolismo , Orthomyxoviridae/genética , Animais , Células Apresentadoras de Antígenos/citologia , Antivirais/metabolismo , Progressão da Doença , Cães , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/metabolismo , Humanos , Sistema Imunitário , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/genética , Fatores de TempoRESUMO
We have determined that, in addition to its receptor-destroying activity, the influenza virus neuraminidase is capable of efficiently forming virus-like particles (VLPs) when expressed individually from plasmid DNA. This observation applies to both human subtypes of neuraminidase, N1 and N2. However, it is not found with every strain of influenza virus. Through gain-of-function and loss-of-function analyses, a critical determinant within the neuraminidase ectodomain was identified that contributes to VLP formation but is not sufficient to accomplish release of plasmid-derived VLPs. This sequence lies on the plasma membrane-proximal side of the neuraminidase globular head. Most importantly, we demonstrate that the antiviral restriction factor tetherin plays a role in determining the strain-specific limitations of release competency. If tetherin is counteracted by small interfering RNA knockdown or expression of the HIV anti-tetherin factor vpu, budding and release capability is bestowed upon an otherwise budding-deficient neuraminidase. These data suggest that budding-competent neuraminidase proteins possess an as-yet-unidentified means of counteracting the antiviral restriction factor tetherin and identify a novel way in which the influenza virus neuraminidase can contribute to virus release.
Assuntos
Antígenos CD/metabolismo , Interações Hospedeiro-Patógeno , Neuraminidase/metabolismo , Orthomyxoviridae/fisiologia , Proteínas Virais/metabolismo , Liberação de Vírus , Linhagem Celular , Proteínas Ligadas por GPI/metabolismo , HumanosRESUMO
Humans infected by the highly pathogenic H5N1 avian influenza viruses (HPAIV) present unusually high concentrations in serum of proinflammatory cytokines and chemokines, which are believed to contribute to the high pathogenicity of these viruses. The hemagglutinins (HAs) of avian influenza viruses preferentially bind to sialic acids attached through α2,3 linkages (SAα2,3) to the terminal galactose of carbohydrates on the host cell surface, while the HAs from human strains bind to α2,6-linked SA (SAα2,6). To evaluate the role of the viral receptor specificity in promoting innate immune responses in humans, we generated recombinant influenza viruses, one bearing the HA and neuraminidase (NA) genes from the A/Vietnam/1203/2004 H5N1 HPAIV in an influenza A/Puerto Rico/8/1934 (A/PR/8/34) backbone with specificity for SAα2,3 and the other a mutant virus (with Q226L and G228S in the HA) with preferential receptor specificity for SAα2,6. Viruses with preferential affinity for SAα2,3 induced higher levels of proinflammatory cytokines and interferon (IFN)-inducible genes in primary human dendritic cells (DCs) than viruses with SAα2,6 binding specificity, and these differences were independent of viral replication, as shown by infections with UV-inactivated viruses. Moreover, human primary macrophages and respiratory epithelial cells showed higher expression of proinflammatory genes after infection with the virus with SAα2,3 affinity than after infection with the virus with SAα2,6 affinity. These data indicate that binding to SAα2,3 by H5N1 HPAIV may be sensed by human cells differently than binding to SAα2,6, inducing an exacerbated innate proinflammatory response in infected individuals.
Assuntos
Imunidade Inata , Virus da Influenza A Subtipo H5N1/imunologia , Virus da Influenza A Subtipo H5N1/fisiologia , Ligação Viral , Animais , Aves , Células Cultivadas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Virus da Influenza A Subtipo H5N1/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Receptores Virais/metabolismo , Recombinação GenéticaRESUMO
Resistance to currently available therapies is a major impediment to the successful treatment of hematological malignancies. Here, we used a model of therapy-resistant B-cell non Hodgkin lymphoma (B-NHL) developed in our laboratory along with primary B-NHL cells to study basic mechanisms of bortezomib activity. In resistant cells and a subset of primary B-NHLs, bortezomib treatment led to stabilization of Bak and subsequent Bak-dependent activation of apoptosis. In contrast to sensitive cells that die strictly by apoptosis, bortezomib was capable of killing resistant cells through activation of apoptosis or caspase-independent mechanism(s) when caspases were pharmacologically inhibited. Our data demonstrate that bortezomib is capable of killing B-NHL cells via multiple mechanisms, regardless of their basal apoptotic potential, and contributes to growing evidence that proteasome inhibitors can act via modulation of B-cell lymphoma 2 (Bcl-2) family proteins. The capacity of bortezomib to act independently of the intrinsic apoptotic threshold of a given B-NHL cell suggests that bortezomib-based therapies could potentially overcome resistance and result in relevant clinical activity in a relapsed/refractory setting.
Assuntos
Antineoplásicos/uso terapêutico , Ácidos Borônicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/uso terapêutico , Western Blotting , Bortezomib , Caspases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Imunoprecipitação , Linfoma de Células B/metabolismo , Prognóstico , Células Tumorais Cultivadas , UbiquitinaçãoRESUMO
BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) is FDA-approved anti-cancer modality for elimination of early disease and palliation in advanced disease. PDT efficacy depends in part on elicitation of a tumor-specific immune response that is dependent on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytolytic potential of CTLs and NK cells is mediated by the ability of these cells to recognize major histocompatibility complex (MHC) class I and MHC class I-related molecules. The MHC class I-related molecules MICA and MICB are induced by oxidative stress and have been reported to activate NK cells and co-stimulate CD8(+) T cells. The purpose of this study was to examine the effect of PDT on tumor cell expression of MHC classes I and II-related molecules in vivo and in vitro. STUDY DESIGN/MATERIALS AND METHODS: Human colon carcinoma Colo205 cells and murine CT26 tumors were treated with 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH)-PDT at various doses. MHC classes I and I-related molecule expression following treatment of Colo205 cells was temporally examined by flow cytometry using antibodies specific for components of MHC class I molecules and by quantitative PCR using specific primers. Expression of MHC class I-related molecules following HPPH-based PDT (HPPH-PDT) of murine tumors was monitored using a chimeric NKG2D receptor. RESULTS: In vitro HPPH-PDT significantly induces MICA in Colo205 cells, but had no effect on MHC class I molecule expression. PDT also induced expression of NKG2D ligands (NKG2DL) following in vivo HPPH-PDT of a murine tumor. Induction of MICA corresponded to increased NK killing of PDT-treated tumor cells. CONCLUSIONS: PDT induction of MICA on human tumor cells and increased expression of NKG2DL by murine tumors following PDT may play a role in PDT induction of anti-tumor immunity. This conclusion is supported by our results demonstrating that tumor cells have increased sensitivity to NK cell lysis following PDT.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Complexo Principal de Histocompatibilidade/efeitos dos fármacos , Fotoquimioterapia/métodos , Animais , Western Blotting , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos BALB C , RNA/análise , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Dengue virus (DENV) is the most prevalent arthropod-borne human virus, able to infect and replicate in human dendritic cells (DCs), inducing their activation and the production of proinflammatory cytokines. However, DENV can successfully evade the immune response in order to produce disease in humans. Several mechanisms of immune evasion have been suggested for DENV, most of them involving interference with type I interferon (IFN) signaling. We recently reported that DENV infection of human DCs does not induce type I IFN production by those infected DCs, impairing their ability to prime naive T cells toward Th1 immunity. In this article, we report that DENV also reduces the ability of DCs to produce type I IFN in response to several inducers, such as infection with other viruses or exposure to Toll-like receptor (TLR) ligands, indicating that DENV antagonizes the type I IFN production pathway in human DCs. DENV-infected human DCs showed a reduced type I IFN response to Newcastle disease virus (NDV), Sendai virus (SeV), and Semliki Forest virus (SFV) infection and to the TLR3 agonist poly(I:C). This inhibitory effect is DENV dose dependent, requires DENV replication, and takes place in DENV-infected DCs as early as 2 h after infection. Expressing individual proteins of DENV in the presence of an IFN-alpha/beta production inducer reveals that a catalytically active viral protease complex is required to reduce type I IFN production significantly. These results provide a new mechanism by which DENV evades the immune system in humans.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/virologia , Vírus da Dengue/imunologia , Vírus da Dengue/patogenicidade , Evasão da Resposta Imune , Interferon Tipo I/biossíntese , Proteínas não Estruturais Virais/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Vírus da Dengue/genética , Vírus da Dengue/fisiologia , Cães , Humanos , Técnicas In Vitro , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/fisiologia , Vírus da Floresta de Semliki/imunologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Receptores Toll-Like/metabolismo , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
Expression of class I human leucocyte antigens (HLA) on the surface of malignant cells is critical for their recognition and destruction by cytotoxic T lymphocytes. Surface expression requires assembly and folding of HLA class I molecules in the endoplasmic reticulum with the assistance of proteins such as Transporter associated with Antigen Processing (TAP) and tapasin. Interferon-gamma induces both TAP and tapasin so dissection of which protein contributes more to HLA class I expression has not been possible previously. In this study, we take advantage of a human melanoma cell line in which TAP can be induced, but tapasin cannot. Interferon-gamma increases TAP protein levels dramatically but HLA class I expression at the cell surface does not increase substantially, indicating that a large increase in peptide supply is not sufficient to increase HLA class I expression. On the other hand, transfection of either allelic form of tapasin (R240 or T240) enhances HLA-B*5001 and HLA-B*5701 antigen expression considerably with only a modest increase in TAP. Together, these data indicate that in the presence of minimal TAP activity, tapasin can promote substantial HLA class I expression at the cell surface.
Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Melanoma/imunologia , Proteínas de Membrana Transportadoras/imunologia , Apresentação de Antígeno/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos HLA-B/metabolismo , Humanos , Interferon gama/imunologia , Proteínas de Neoplasias/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
OBJECTIVE: The ability to sustain and grow portions of human tumors as xenografts in SCID mice provides a valuable preclinical opportunity to test the response of human tumors to treatments, both individually and in combination. Using this model, our laboratory has previously demonstrated that the growth of several human adenocarcinomas can be inhibited by Apo2L/TRAIL. Apo2L/TRAIL triggers apoptosis in many types of tumor cells, and when combined with various chemotherapeutic agents results in enhanced inhibition of tumor growth in many xenograft models. METHODS: To gain further insight into the antitumor potential of Apo2L/TRAIL in combination with chemotherapy, we compared the responses of 2 human colon adenocarcinomas, both of which were sensitive to CPT-11 while one was sensitive and the other comparatively resistant to Apo2L/TRAIL. RESULTS: In both cases, a greater degree of growth inhibition was achieved when these agents were used in combination. Western blot analysis demonstrated that in the Apo2L/TRAIL-sensitive tumor total cellular expression of Apo2L/TRAIL death receptors (DR4 and DR5) as well as protein expression of the pro-apoptotic molecule BAX were higher and the anti-apoptotic molecule Bcl-2 was lower in comparison to the Apo2L/TRAIL-resistant tumor. CONCLUSION: These results indicate that both Apo2L/TRAIL-sensitive and -resistant colon tumors will respond to a combination of CPT-11 and Apo2L/TRAIL and predict that this will be useful in the treatment of human colon cancers in a clinical setting.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Camptotecina/análogos & derivados , Neoplasias do Colo/terapia , Terapia Genética , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Adenocarcinoma/genética , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Animais , Apoptose/fisiologia , Western Blotting , Camptotecina/uso terapêutico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Terapia Combinada , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Irinotecano , Camundongos , Camundongos SCID , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Inibidores da Topoisomerase I , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The ability for serially homologous structures to acquire a separate identity has been primarily investigated for structures dependent on Hox gene input but is still incompletely understood in other systems. The fore and hindwings of butterflies are serially homologous structures as are the serially homologous eyespots that can decorate each of these wings. Eyespots can vary in number between fore and hindwings of the same individual and mutations of large effect can control the total number of eyespots that each of the wings displays. Here we investigate the genetics of a new spontaneous color pattern mutation, Missing, that alters eyespot number in the nymphalid butterfly, Bicyclus anynana. We further test the interaction of Missing with a previously described mutation, Spotty, describe the developmental stage affected by Missing, and test whether Missing is a mutant variant of the gene Distal-less via a linkage association study. RESULTS: Missing removes or greatly reduces the size of two of the hindwing eyespots from the row of seven eyespots, with no detectable effect on the rest of the wing pattern. Offspring carrying a single Missing allele display intermediate sized eyespots at these positions. Spotty has the opposite effect of Missing, i.e., it introduces two extra eyespots in homologous wing positions to those affected by Missing, but on the forewing. When Missing is combined with Spotty the size of the two forewing eyespots decreases but the size of the hindwing spots stays the same, suggesting that these two mutations have a combined effect on the forewing such that Missing reduces eyespot size when in the presence of a Spotty mutant allele, but that Spotty has no effect on the hindwing. Missing prevents the complete differentiation of two of the eyespot foci on the hindwing. We found no evidence for any linkage between the Distal-less and Missing genes. CONCLUSION: The spontaneous mutation Missing controls the differentiation of the signaling centers of a subset of the serial homologous eyespots present on both the fore and the hindwing in a dose-dependent fashion. The effect of Missing on the forewing, however, is only observed when the mutation Spotty introduces additional eyespots on this wing. Spotty, on the other hand, controls the differentiation of eyespot centers only on the forewing. Spotty, unlike Missing, may be under Ubx gene regulation, since it affects a subset of eyespots on only one of the serially homologous wings.
Assuntos
Borboletas/genética , Mutação , Pigmentação/genética , Asas de Animais/anatomia & histologia , Animais , Borboletas/anatomia & histologia , Cruzamentos Genéticos , Feminino , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Caracteres SexuaisRESUMO
La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of "blue eye disease", causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/ß.
Assuntos
Interferon Tipo I/antagonistas & inibidores , Rubulavirus/imunologia , Fator de Transcrição STAT2/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Humanos , Ligação Proteica , Transporte Proteico , SuínosRESUMO
We previously demonstrated that ectodomain residue Asp286 in N2 neuraminidase (NA; Asp268 in N1 NA) present in budding-capable NA proteins contributes to productive NA plasma membrane transport partly by mediating escape from tetherin restriction [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Budding-incapable NA proteins contain a G at this position and either co-expression of human immunodeficiency virus type 1 vpu or siRNA-mediated depletion of tetherin rescued budding capabilities in these proteins [Yondola MA, Fernandes F, Belicha-Villanueva A, Uccelini M, Gao Q, Carter C, et al. (2011). Budding capability of the influenza virus neuraminidase can be modulated by tetherin. J Virol, 85, 2480-2491]. Furthermore, replacement of D286 with G in budding-capable NA proteins caused loss of function, preventing release of NA virus-like particles (VLPs). Here, we show that mutation of this residue specifically modulates the ability of NA to escape tetherin restriction at the plasma membrane and results in virus attenuation in vivo. Based on immunogold electron microscopy and co-immunoprecipitation assays, both NAD286-containing and NAD286G-containing proteins associated with tetherin in the endoplasmic reticulum (ER). However, the NAD286G loss-of-function mutant also associated with the host factor outside the ER and in plasma-membrane-localized VLPs as visualized using immunogold electron microscopy. We conclude that the presence of aspartate at residue 286 liberates NA from tetherin-dependent restriction upon exit from the ER compartment thus preventing restriction at the plasma membrane. Underscoring the importance of these observations, knockdown of tetherin resulted in a 1-1.5 log increase in influenza virus growth. Additionally, the loss-of-function mutation conferred attenuation in a mouse model of influenza infection as evidenced by a 5-fold increase in LD50 and increases in either percent survival or time to death dependent on the administered dose in vivo.
Assuntos
Antígenos CD/metabolismo , Vírus da Influenza A/patogenicidade , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/virologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Antígenos CD/genética , Células COS , Chlorocebus aethiops , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Estrutura Terciária de Proteína , Proteínas Virais/genética , Vírion/fisiologiaRESUMO
Assembly of major histocompatibility complex (MHC) class I molecules with peptide in the endoplasmic reticulum requires the assistance of tapasin. Alternative splicing, which is known to regulate many genes, has been reported for tapasin only in the context of mutations. Here, we report on an alternate splice form of tapasin (tpsnΔEx3) derived from a human melanoma cell line that does not appear to be caused by mutations. Excision of exon 3 results in deletion of amino acids 70 to 156 within the beta barrel region, but the membrane proximal Ig domain, the transmembrane domain, and cytoplasmic tail of tapasin are intact. Introduction of tpsnΔEx3 into a tapasin-deficient cell line does not restore MHC class I expression at the cell surface. Similar to a previously described tapasin mutant (tpsnΔN50), tpsnΔEx3 interacts with TAP. Therefore, we used these altered forms of tapasin to test the importance of MHC class I interaction with TAP. In the presence of wild-type tapasin, transfection of tpsnΔN50, but not tpsnΔEx3, reduced MHC class I expression at the cell surface likely due its ability to compete MHC class I molecules from TAP. Together these findings suggest that tumor cells may contain alternate splice forms of tapasin which may regulate MHC class I antigen presentation.
Assuntos
Processamento Alternativo/imunologia , Melanoma/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica/imunologia , Isoformas de Proteínas/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Complexos Multiproteicos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Estrutura Terciária de Proteína/genética , Deleção de Sequência/genética , Transgenes/genéticaRESUMO
The expression of major histocompatibility complex (MHC) class I molecules on the cell surface is critical for recognition by cytotoxic T lymphocytes (CTL). This recognition event leads to destruction of cells displaying MHC class I-viral peptide complexes or cells displaying MHC class I-mutant peptide complexes. Before they can be transported to the cell surface, MHC class I molecules must associate with their peptide ligand in the endoplasmic reticulum (ER) of the cell. Within the ER, numerous proteins assist in the appropriate assembly and folding of MHC class I molecules. These include the heterodimeric transporter associated with antigen processing (TAP1 and TAP2), the heterodimeric chaperone-oxidoreductase complex of tapasin and ERp57 and the general ER chaperones calreticulin and calnexin. Each of these accessory proteins has a well-defined role in antigen presentation by MHC class I molecules. However, alternate splice forms of MHC class I heavy chains, TAP and tapasin, have been reported suggesting additional complexity to the picture of antigen presentation. Here, we review the importance of these different accessory proteins and the progress in our understanding of alternate splicing in antigen presentation.
Assuntos
Processamento Alternativo/imunologia , Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Animais , Apresentação de Antígeno/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologiaRESUMO
Dengue virus encodes several interferon antagonists. Among these the NS5 protein binds STAT2, a necessary component of the type I interferon signaling pathway, and targets it for degradation. We now demonstrate that the ability of dengue NS5 to associate with and degrade STAT2 is species specific. Thus, NS5 is able to bind and degrade human STAT2, but not mouse STAT2. This difference was exploited to demonstrate, absent manipulation of the viral genome, that NS5-mediated IFN antagonism is essential for efficient virus replication. Moreover, we demonstrate that differences in NS5 mediated binding and degradation between human and mouse STAT2 maps to a region within the STAT2 coiled-coil domain. By using STAT2(-/-) mice, we also demonstrate that mouse STAT2 restricts early dengue virus replication in vivo. These results suggest that overcoming this restriction through transgenic mouse technology may help in the development of a long-sought immune-competent mouse model of dengue virus infection.
Assuntos
Vírus da Dengue/fisiologia , Fator de Transcrição STAT2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Cricetinae , Vírus da Dengue/metabolismo , Humanos , Interferons/antagonistas & inibidores , Interferons/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT2/química , Fator de Transcrição STAT2/genética , Transdução de Sinais , Especificidade da Espécie , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Four combinations of drugs--ketamine-xylazine, ketamine-xylazine-acepromazine (KXA), ketamine-xylazine-buprenorphine, and ketamine-xylazine-carprofen--were compared for their ability to produce anesthesia in BALB/c mice. Induction time, anesthetic duration, blood pressure, pulse rate, and time to recovery were recorded. The anesthesia induced by each anesthetic combination was assessed by using reflex responses to standardized stimuli. The KXA combination produced stable physiologic parameters and was associated with the longest duration of anesthesia (40 +/- 8 min); immobility was produced in all other groups (38 +/- 5 min), but a surgical plane of anesthesia could not be confirmed. All anesthetic protocols produced significant hypotension. No deaths occurred. We recommend KXA as a safe and reliable anesthetic for mice requiring a surgical plane of anesthesia.