RESUMO
Nanodiamonds were combined with niosome, and resulting formulations were named as nanodiasomes, which were evaluated in terms of physicochemical features, cellular internalization, cell viability and transfection efficiency both in in vitro and in in vivo conditions. Such parameters were analyzed at 4 and 25 °C, and at 15 and 30 days after their elaboration. Nanodiasomes showed a particle size of 128 nm that was maintained over time inside the ± 10% of deviation, unless after 30 days of storage at 25 °C. Something similar occurred with the initial zeta potential value, 35.2 mV, being both formulations more stable at 4 °C. The incorporation of nanodiamonds into niosomes resulted in a 4-fold increase of transfection efficiency that was maintained over time at 4 and 25 °C. In vivo studies reported high transgene expression of nanodiasomes after subretinal and intravitreal administration in mice, when injected freshly prepared and after 30 days of storage at 4 °C.
Assuntos
Nanodiamantes , Ratos , Camundongos , Animais , Ratos Sprague-Dawley , Linhagem Celular , Retina/metabolismo , Lipossomos , LipídeosRESUMO
Alphaviruses are emerging positive-stranded RNA viruses which replicate and transcribe their genomes in membranous organelles formed in the cell cytoplasm. The nonstructural protein 1 (nsP1) is responsible for viral RNA capping and gates the replication organelles by assembling into monotopic membrane-associated dodecameric pores. The capping pathway is unique to Alphaviruses; beginning with the N7 methylation of a guanosine triphosphate (GTP) molecule, followed by the covalent linkage of an m7GMP group to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate RNA. Here, we provide structural snapshots of different stages of the reaction pathway showing how nsP1 pores recognize the substrates of the methyl-transfer reaction, GTP and S-adenosyl methionine (SAM), how the enzyme reaches a metastable postmethylation state with SAH and m7GTP in the active site, and the subsequent covalent transfer of m7GMP to nsP1 triggered by the presence of RNA and postdecapping reaction conformational changes inducing the opening of the pore. In addition, we biochemically characterize the capping reaction, demonstrating specificity for the RNA substrate and the reversibility of the cap transfer resulting in decapping activity and the release of reaction intermediates. Our data identify the molecular determinants allowing each pathway transition, providing an explanation for the need for the SAM methyl donor all along the pathway and clues about the conformational rearrangements associated to the enzymatic activity of nsP1. Together, our results set ground for the structural and functional understanding of alphavirus RNA-capping and the design of antivirals.
Assuntos
Alphavirus , Febre de Chikungunya , Alphavirus/genética , Antivirais/farmacologia , Guanosina Trifosfato/metabolismo , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas não Estruturais Virais/genética , Replicação ViralRESUMO
AIMS: Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. METHODS AND RESULTS: We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. CONCLUSION: Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.
Assuntos
Alphainfluenzavirus/patogenicidade , Sistema de Condução Cardíaco/virologia , Miocardite/virologia , Infecções por Orthomyxoviridae/virologia , Animais , Conexinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Matriz Extracelular/metabolismo , Matriz Extracelular/virologia , Feminino , Fibrose , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/patologia , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Alphainfluenzavirus/genética , Alphainfluenzavirus/crescimento & desenvolvimento , Cinética , Pulmão/virologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mutação , Miocardite/metabolismo , Miocardite/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/virologia , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Ramos Subendocárdicos/metabolismo , Ramos Subendocárdicos/virologia , Carga Viral , Virulência , Replicação Viral , Proteína alfa-5 de Junções ComunicantesRESUMO
Influenza virus infection increases the methylation of lysine 79 of histone 3 catalyzed by the Dot1L enzyme. The role of Dot1L against infections was highlighted by an increase of influenza A and vesicular stomatitis virus replication in Dot1L-inhibited cells mediated by a decreased antiviral response. Interferon-beta (IFN-ß) reporter assays indicate that Dot1L is involved in the control of retinoic acid-inducible geneI protein (RIG-I) signaling. Accordingly, Dot1L inhibition decreases the IFN-ß promoter stimulation and RIG-I- mitochondria-associated viral sensor (RIG-I-MAVS) association upon viral infection. Replication of an influenza A virus lacking NS1 (delNS1), incapable of counteracting the antiviral response, is not affected by Dot1L inhibition. Consequently, RIG-I-MAVS association and nuclear factor-ï«B (NF-κïï© nuclear translocation, are not affected by the Dot1L inhibition in delNS1 infected cells. Restoration of NS1 expression in trans also reinstated Dot1L as a regulator of the RIG-I-dependent signaling in delNS1 infections. Interferon-inducible E3 ligase tripartite motif-containing protein 25 (TRIM25) expression increases in influenza virus infected cells, but Dot1L inhibition reduces both the TRIM25 expression and TRIM25 protein levels. TRIM25 overexpression reverses the defective innate response mediated by Dot1L inhibition elicited upon virus infection or by overexpression of RIG-I signaling intermediates. Thus, TRIM25 is a control point of the RIG-I recognition pathway controlled by Dot1L and may have a general role in RNA viruses recognized by the RIG-I sensor.
Assuntos
Histona Metiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Vírus da Influenza A/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Histona Metiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Imunidade Inata/imunologia , Vírus da Influenza A/genética , Transdução de Sinais/fisiologia , Proteínas não Estruturais Virais/metabolismoRESUMO
The vRNAs of influenza A viruses contain 12 and 13 nucleotide-long sequences at their 3' and 5' termini respectively that are highly conserved and constitute the vRNA promoter. These sequences and the next three segment-specific nucleotides show inverted partial complementarity and are followed by several unpaired nucleotides of poorly characterized function at the 3' end. We have performed systematic point-mutations at the segment-specific nucleotides 15-18 of the 3'-end of a NS-like vRNA segment. All NS-like vRNAs containing mutations at position 15, and some at positions 16-18 showed reduced transcription/replication efficiency in a transfection/infection system. In addition, the replication of recombinant viruses containing mutations at position 15 was impaired both in single and multi-cycle experiments. This reduction was the consequence of a decreased expression of the NS segment. The data indicate that NS1 plays a role in the transcription/replication of its own segment, which elicits a global defect on virus replication.
Assuntos
Regiões 3' não Traduzidas/genética , Vírus da Influenza A/fisiologia , Replicação Viral , Células A549 , Animais , Cães , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Vírus da Influenza A/genética , Células Madin Darby de Rim Canino , Mutação , RNA Viral/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Characterization of a pandemic 2009 H1N1 influenza virus isolated from a fatal case patient (F-IAV), showed the presence of three different mutations; potential determinants of its high pathogenicity that were located in the polymerase subunits (PB2 A221T and PA D529N) and the hemagglutinin (HA S110L). Recombinant viruses containing individually or in combination the polymerase mutations in the backbone of A/California/04/09 (CAL) showed that PA D529N was clearly involved in the increased pathogenicity of the F-IAV virus. Here, we have evaluated the contribution of HA S110L to F-IAV pathogenicity, through introduction of this point mutation in CAL recombinant virus (HA mut). The HA S110L protein has similar pH stability, comparable mobility, and entry properties both in human and mouse cultured cells that wild type HA. The change HA S110L leads to a non-significant trend to reduce the replication capacity of influenza virus in tissue culture, and HA mut is better neutralized than CAL virus by monoclonal and polyclonal antibodies against HA from CAL strain. In addition, recombinant viruses containing HA S110L alone or in combination with polymerase mutations considerably increased the LD50 in infected mice. Characterization of the lungs of HA mut infected animals showed reduced lung damage and inflammation compared with CAL infected mice. Accordingly, lower virus replication, decreased presence in bronchioli and parenchyma and lower leukocytes and epithelial infected cells were found in the lungs of HA mut-infected animals. Our results indicate that, mutation HA S110L constitutes a determinant of attenuation and suggest that its interaction with components of the respiratory tract mucus and lectins, that play an important role on influenza virus outcome, may constitute a physical barrier impeding the infection of the target cells, thus compromising the infection outcome.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/virologia , Pulmão/patologia , Mutação/genética , Infecções por Orthomyxoviridae/virologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunidade , Vírus da Influenza A Subtipo H1N1/patogenicidade , Pulmão/virologia , Camundongos , Virulência , Internalização do Vírus , Replicação ViralRESUMO
Influenza virus stablishes a network of virus-host functional interactions, which depends on chromatin dynamic and therefore on epigenetic modifications. Using an unbiased search, we analyzed the epigenetic changes at DNA methylation and post-translational histone modification levels induced by the infection. DNA methylation was unaltered, while we found a general decrease on histone acetylation, which correlates with transcriptional inactivation and may cooperate with the impairment of cellular transcription that causes influenza virus infection. A particular increase in H3K79 methylation was observed and the use of an inhibitor of the specific H3K79 methylase, Dot1L enzyme, or its silencing, increased influenza virus replication. The antiviral response was reduced in conditions of Dot1L downregulation, since decreased nuclear translocation of NF-kB complex, and IFN-ß, Mx1 and ISG56 expression was detected. The data suggested a control of antiviral signaling by methylation of H3K79 and consequently, influenza virus replication was unaffected in IFN pathway-compromised, Dot1L-inhibited cells. H3K79 methylation also controlled replication of another potent interferon-inducing virus such as vesicular stomatitis virus, but did not modify amplification of respiratory syncytial virus that poorly induces interferon signaling. Epigenetic methylation of H3K79 might have an important role in controlling interferon-induced signaling against viral pathogens.
Assuntos
Metilação de DNA , Epigênese Genética , Código das Histonas , Interações Hospedeiro-Patógeno , Orthomyxoviridae/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linhagem Celular Tumoral , Cães , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Interferon beta/metabolismo , Células Madin Darby de Rim Canino , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , NF-kappa B/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição/metabolismo , Replicação ViralRESUMO
Influenza A virus (IAV) infection can be severe or even lethal in toddlers, the elderly and patients with certain medical conditions. Infection of apparently healthy individuals nonetheless accounts for many severe disease cases and deaths, suggesting that viruses with increased pathogenicity co-circulate with pandemic or epidemic viruses. Looking for potential virulence factors, we have identified a polymerase PA D529N mutation detected in a fatal IAV case, whose introduction into two different recombinant virus backbones, led to reduced defective viral genomes (DVGs) production. This mutation conferred low induction of antiviral response in infected cells and increased pathogenesis in mice. To analyze the association between low DVGs production and pathogenesis in humans, we performed a genomic analysis of viruses isolated from a cohort of previously healthy individuals who suffered highly severe IAV infection requiring admission to Intensive Care Unit and patients with fatal outcome who additionally showed underlying medical conditions. These viruses were compared with those isolated from a cohort of mild IAV patients. Viruses with fewer DVGs accumulation were observed in patients with highly severe/fatal outcome than in those with mild disease, suggesting that low DVGs abundance constitutes a new virulence pathogenic marker in humans.
Assuntos
Genoma Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/virologia , Infecções por Orthomyxoviridae/virologia , Replicação Viral/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Virus da Influenza A Subtipo H5N1/patogenicidade , Masculino , Camundongos , Pessoa de Meia-Idade , Infecções por Orthomyxoviridae/genética , Virulência/genética , Adulto JovemRESUMO
The replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified, and analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive or negative polarity. Their generation in vivo was strictly dependent on the polymerase activity; they contained mainly negative-polarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Sequência de Bases , Linhagem Celular , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Genoma Viral , Humanos , Imageamento Tridimensional , Substâncias Macromoleculares , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutagênese Sítio-Dirigida , Sondas RNA/genética , RNA Viral/ultraestrutura , Transcrição Gênica , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
During thymocyte maturation, enhancers of genes encoding for TCRdelta (Tcrd) and TCRalpha (Tcra), Edelta(8), and Ealpha, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an Ealpha fragment, Talpha1-Talpha2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire Ealpha or Talpha1-Talpha4. These experiments indicated that Talpha3-Talpha4 collaborates with factors bound to Talpha1-Talpha2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on Talpha1-Talpha2 explained the molecular basis for requirement of intact Talpha2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of Ealpha, EalphaMC, in which Edelta myb and runx sites have been substituted for Talpha2 runx and ets sites, that argues against the notion of a requirement for strict Ealpha enhanceosome structure for function. EalphaMC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an Ealpha enhanceosome are rather flexible. Activation of V(D)J recombination by EalphaMC during thymocyte development resulted, however, to be premature and indistinguishable from that of Talpha1-Talpha2. These results indicate that Talpha3-Talpha4 itself is not sufficient to impart a developmental delay to a chimeric "early" enhancer, and indicate the need for functional collaboration between Talpha2 runx/ets sites binding proteins and proteins bound to Talpha3-Talpha4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on Ealpha might represent a more flexible form of information processing during thymocyte development.
Assuntos
Elementos Facilitadores Genéticos , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T/genética , Complexos Multiproteicos/genética , Nucleoproteínas/química , Multimerização Proteica , Nucleoproteínas/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta , Timo/citologiaRESUMO
The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. As PB2 is a relevant host-range determinant we expressed a TAP-tagged PB2 in human cells and isolated intracellular complexes. Alpha-importin was identified as a PB2-associated factor by proteomic analyses. To study the relevance of this interaction for virus replication we mutated the PB2 NLS and analysed the phenotype of mutant subunits, polymerase complexes and RNPs. While mutant PB2 proteins showed reduced nuclear accumulation, they formed polymerase complexes normally when co expressed with PB1 and PA. However, mutant RNPs generated with a viral CAT replicon showed up to hundred-fold reduced CAT accumulation. Rescue of nuclear localisation of mutant PB2 by insertion of an additional SV40 TAg-derived NLS did not revert the mutant phenotype of RNPs. Furthermore, determination of recombinant RNP accumulation in vivo indicated that PB2 NLS mutations drastically reduced virus RNA replication. These results indicate that, above and beyond its role in nuclear accumulation, PB2 interaction with alpha-importins is required for virus RNA replication. To ascertain whether PB2-alpha-importin binding could contribute to the adaptation of H5N1 avian viruses to man, their association in vivo was determined. Human alpha importin isoforms associated efficiently to PB2 protein of an H3N2 human virus but bound to diminished and variable extents to PB2 from H5N1 avian or human strains, suggesting that the function of alpha importin during RNA replication is important for the adaptation of avian viruses to the human host.
Assuntos
Orthomyxoviridae/enzimologia , Orthomyxoviridae/fisiologia , Subunidades Proteicas/metabolismo , RNA Viral/biossíntese , Proteínas Virais/metabolismo , Replicação Viral , alfa Carioferinas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Espaço Intracelular/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Sinais de Localização Nuclear/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/metabolismo , Especificidade da Espécie , Proteínas Virais/química , alfa Carioferinas/químicaRESUMO
The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.
Assuntos
Orthomyxoviridae/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , RNA Helicases DEAD-box/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The TCR delta enhancer (Edelta) and TCR alpha enhancer (Ealpha) play critical roles in the temporal and lineage-specific control of V(D)J recombination and transcription at the TCR alphadelta locus, working as a developmental switch controlling a transition from TCR delta to TCR alpha activity during thymocyte development. Previous experiments using a transgenic reporter substrate revealed that substitution of the 116-bp minimal Ealpha, denoted Talpha1-Talpha2, for the entire 1.4-kb Ealpha led to a premature activation of V(D)J recombination. This suggested that binding sites outside of Talpha1-Talpha2 are critical for the strict developmental regulation of TCR alpha rearrangement. We have further analyzed Ealpha to better understand the mechanisms responsible for appropriate developmental regulation in vivo. We found that a 275-bp Ealpha fragment, denoted Talpha1-Talpha4, contains all binding sites required for proper developmental regulation in vivo. This suggests that developmentally appropriate enhancer activation results from a functional interaction between factors bound to Talpha1-Talpha2 and Talpha3-Talpha4. In support of this, EMSAs reveal the formation of a large enhanceosome complex that reflects the cooperative assembly of proteins bound to both Talpha1-Talpha2 and Talpha3-Talpha4. Our data suggest that enhanceosome assembly is critical for developmentally appropriate activation of Ealpha in vivo, and that transcription factors, Sp1 and pCREB, may play unique roles in this process.