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1.
J Immunol ; 210(12): 1925-1937, 2023 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-37098890

RESUMEN

COVID-19 has accounted for more than 6 million deaths worldwide. Bacillus Calmette-Guérin (BCG), the existing tuberculosis vaccine, is known to induce heterologous effects over other infections due to trained immunity and has been proposed to be a potential strategy against SARS-CoV-2 infection. In this report, we constructed a recombinant BCG (rBCG) expressing domains of the SARS-CoV-2 nucleocapsid and spike proteins (termed rBCG-ChD6), recognized as major candidates for vaccine development. We investigated whether rBCG-ChD6 immunization followed by a boost with the recombinant nucleocapsid and spike chimera (rChimera), together with alum, provided protection against SARS-CoV-2 infection in K18-hACE2 mice. A single dose of rBCG-ChD6 boosted with rChimera associated with alum elicited the highest anti-Chimera total IgG and IgG2c Ab titers with neutralizing activity against SARS-CoV-2 Wuhan strain when compared with control groups. Importantly, following SARS-CoV-2 challenge, this vaccination regimen induced IFN-γ and IL-6 production in spleen cells and reduced viral load in the lungs. In addition, no viable virus was detected in mice immunized with rBCG-ChD6 boosted with rChimera, which was associated with decreased lung pathology when compared with BCG WT-rChimera/alum or rChimera/alum control groups. Overall, our study demonstrates the potential of a prime-boost immunization system based on an rBCG expressing a chimeric protein derived from SARS-CoV-2 to protect mice against viral challenge.


Asunto(s)
COVID-19 , Mycobacterium bovis , Animales , Ratones , Vacuna BCG/genética , Proteínas Recombinantes de Fusión/genética , SARS-CoV-2 , Vacunas Sintéticas , COVID-19/prevención & control , Mycobacterium bovis/genética
2.
Nucleic Acids Res ; 48(9): 5081-5093, 2020 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-32313955

RESUMEN

Flaviviruses, including dengue virus and Zika virus, contain a single-stranded positive sense RNA genome that encodes viral proteins essential for replication and also serves as the template for new genome synthesis. As these processes move in opposite directions along the genome, translation must be inhibited at a defined point following infection to clear the template of ribosomes to allow efficient replication. Here, we demonstrate in vitro and in cell-based assays that the viral RNA polymerase, NS5, inhibits translation of the viral genome. By reconstituting translation in vitro using highly purified components, we show that this translation block occurs at the initiation stage and that translation inhibition depends on NS5-RNA interaction, primarily through association with the 5' replication promoter region. This work supports a model whereby expression of a viral protein signals successful translation of the infecting genome, prompting a switch to a ribosome depleted replication-competent form.


Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Genoma Viral , Biosíntesis de Proteínas , ARN Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Chlorocebus aethiops , Virus del Dengue/enzimología , Iniciación de la Cadena Peptídica Traduccional , ARN Viral/química , Células Vero , Replicación Viral , Virus Zika/enzimología , Virus Zika/fisiología
3.
J Immunol ; 202(9): 2671-2681, 2019 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-30894428

RESUMEN

Brucella abortus is a facultative intracellular bacterium that causes brucellosis, a prevalent zoonosis that leads to abortion and infertility in cattle, and undulant fever, debilitating arthritis, endocarditis, and meningitis in humans. Signaling pathways triggered by B. abortus involves stimulator of IFN genes (STING), which leads to production of type I IFNs. In this study, we evaluated the pathway linking the unfolded protein response (UPR) and the endoplasmic reticulum-resident transmembrane molecule STING, during B. abortus infection. We demonstrated that B. abortus infection induces the expression of the UPR target gene BiP and XBP1 in murine macrophages through a STING-dependent pathway. Additionally, we also observed that STING activation was dependent on the bacterial second messenger cyclic dimeric GMP. Furthermore, the Brucella-induced UPR is crucial for induction of multiple molecules linked to type I IFN signaling pathway, such as IFN-ß, IFN regulatory factor 1, and guanylate-binding proteins. Furthermore, IFN-ß is also important for the UPR induction during B. abortus infection. Indeed, IFN-ß shows a synergistic effect in inducing the IRE1 axis of the UPR. In addition, priming cells with IFN-ß favors B. abortus survival in macrophages. Moreover, Brucella-induced UPR facilitates bacterial replication in vitro and in vivo. Finally, these results suggest that B. abortus-induced UPR is triggered by bacterial cyclic dimeric GMP, in a STING-dependent manner, and that this response supports bacterial replication. In summary, association of STING and IFN-ß signaling pathways with Brucella-induced UPR unravels a novel link between innate immunity and endoplasmic reticulum stress that is crucial for bacterial infection outcome.


Asunto(s)
Brucella abortus/fisiología , Brucelosis/inmunología , Interacciones Huésped-Patógeno/inmunología , Proteínas de la Membrana/inmunología , Nucleótidos Cíclicos/inmunología , Respuesta de Proteína Desplegada/inmunología , Animales , Brucelosis/genética , Interacciones Huésped-Patógeno/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Nucleótidos Cíclicos/genética , Transducción de Señal/genética , Transducción de Señal/inmunología
4.
Nucleic Acids Res ; 46(10): 5269-5285, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29554348

RESUMEN

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed during the cell-intrinsic immune response to viral infection. IFIT1 inhibits translation by binding directly to the 5' end of foreign RNAs, particularly those with non-self cap structures, precluding the recruitment of the cap-binding eukaryotic translation initiation factor 4F and ribosome recruitment. The presence of IFIT1 imposes a requirement on viruses that replicate in the cytoplasm to maintain mechanisms to avoid its restrictive effects. Interaction of different IFIT family members is well described, but little is known of the molecular basis of IFIT association or its impact on function. Here, we reconstituted different complexes of IFIT1, IFIT2 and IFIT3 in vitro, which enabled us to reveal critical aspects of IFIT complex assembly. IFIT1 and IFIT3 interact via a YxxxL motif present in the C-terminus of each protein. IFIT2 and IFIT3 homodimers dissociate to form a more stable heterodimer that also associates with IFIT1. We show for the first time that IFIT3 stabilizes IFIT1 protein expression, promotes IFIT1 binding to a cap0 Zika virus reporter mRNA and enhances IFIT1 translation inhibition. This work reveals molecular aspects of IFIT interaction and provides an important missing link between IFIT assembly and function.


Asunto(s)
Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/genética , Cromatografía en Gel , Genes Reporteros , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Virus Zika/genética
5.
J Virol ; 92(10)2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29514900

RESUMEN

Dengue virus (DV) infection can cause either a self-limiting flu-like disease or a threatening hemorrhage that may evolve to shock and death. A variety of cell types, such as dendritic cells, monocytes, and B cells, can be infected by DV. However, despite the role of T lymphocytes in the control of DV replication, there remains a paucity of information on possible DV-T cell interactions during the disease course. In the present study, we have demonstrated that primary human naive CD4+ and CD8+ T cells are permissive for DV infection. Importantly, both T cell subtypes support viral replication and secrete viable virus particles. DV infection triggers the activation of both CD4+ and CD8+ T lymphocytes, but preactivation of T cells reduces the susceptibility of T cells to DV infection. Interestingly, the cytotoxicity-inducing protein granzyme A is highly secreted by human CD4+ but not CD8+ T cells after exposure to DV in vitro Additionally, using annexin V and polycaspase assays, we have demonstrated that T lymphocytes, in contrast to monocytes, are resistant to DV-induced apoptosis. Strikingly, both CD4+ and CD8+ T cells were found to be infected with DV in acutely infected dengue patients. Together, these results show that T cells are permissive for DV infection in vitro and in vivo, suggesting that this cell population may be a viral reservoir during the acute phase of the disease.IMPORTANCE Infection by dengue virus (DV) causes a flu-like disease that can evolve to severe hemorrhaging and death. T lymphocytes are important cells that regulate antibody secretion by B cells and trigger the death of infected cells. However, little is known about the direct interaction between DV and T lymphocytes. Here, we show that T lymphocytes from healthy donors are susceptible to infection by DV, leading to cell activation. Additionally, T cells seem to be resistant to DV-induced apoptosis, suggesting a potential role as a viral reservoir in humans. Finally, we show that both CD4+ and CD8+ T lymphocytes from acutely infected DV patients are infected by DV. Our results raise new questions about DV pathogenesis and vaccine development.


Asunto(s)
Apoptosis/inmunología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Virus del Dengue/inmunología , Dengue/inmunología , Activación de Linfocitos/inmunología , Adolescente , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Dengue/virología , Virus del Dengue/fisiología , Femenino , Granzimas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Replicación Viral/inmunología , Adulto Joven
6.
J Biol Chem ; 290(10): 5991-6002, 2015 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-25605733

RESUMEN

Vaccinia virus (VACV) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κB (NF-κB). VACV protein A49 prevents translocation of NF-κB to the nucleus by sequestering cellular ß-TrCP, a protein required for the degradation of the inhibitor of κB. A49 does not share overall sequence similarity with any protein of known structure or function. We solved the crystal structure of A49 from VACV Western Reserve to 1.8 Å resolution and showed, surprisingly, that A49 has the same three-dimensional fold as Bcl-2 family proteins despite lacking identifiable sequence similarity. Whereas Bcl-2 family members characteristically modulate cellular apoptosis, A49 lacks a surface groove suitable for binding BH3 peptides and does not bind proapoptotic Bcl-2 family proteins Bax or Bak. The N-terminal 17 residues of A49 do not adopt a single well ordered conformation, consistent with their proposed role in binding ß-TrCP. Whereas pairs of A49 molecules interact symmetrically via a large hydrophobic surface in crystallo, A49 does not dimerize in solution or in cells, and we propose that this hydrophobic interaction surface may mediate binding to a yet undefined cellular partner. A49 represents the eleventh VACV Bcl-2 family protein and, despite these proteins sharing very low sequence identity, structure-based phylogenetic analysis shows that all poxvirus Bcl-2 proteins are structurally more similar to each other than they are to any cellular or herpesvirus Bcl-2 proteins. This is consistent with duplication and diversification of a single BCL2 family gene acquired by an ancestral poxvirus.


Asunto(s)
Inmunidad Innata/genética , Filogenia , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Virales/química , Apoptosis/genética , Cristalografía por Rayos X , Células HEK293 , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Conformación Proteica , Pliegue de Proteína , Vaccinia/genética , Vaccinia/virología , Virus Vaccinia/química , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Proteínas Virales/genética
7.
PLoS Pathog ; 9(2): e1003183, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23468625

RESUMEN

The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKß causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase ß-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished ß-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.


Asunto(s)
Imitación Molecular , FN-kappa B/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , Virus Vaccinia/patogenicidad , Virus de la Viruela/patogenicidad , Proteínas con Repetición de beta-Transducina/metabolismo , Animales , Línea Celular , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Evasión Inmune , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Ubiquitina-Proteína Ligasas/genética , Virus Vaccinia/genética , Virus Vaccinia/inmunología , Virus de la Viruela/genética , Virus de la Viruela/inmunología , Virulencia , Proteínas con Repetición de beta-Transducina/genética
8.
Phytother Res ; 29(10): 1509-15, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26094613

RESUMEN

Several plant species are used in Brazil to treat inflammatory diseases and associated conditions. TNF-α plays a pivotal role on inflammation, and several plant extracts have been assayed against this target, both in vitro and in vivo. The effect of 11 Brazilian medicinal plants on TNF-α release by LPS-activated THP-1 cells was evaluated. The plant materials were percolated with different solvents to afford 15 crude extracts, whose effect on TNF-α release was determined by ELISA. Among the evaluated extracts, only Jacaranda caroba (Bignoniaceae) presented strong toxicity to THP-1 cells. Considering the 14 non-toxic extracts, TNF-α release was significantly reduced by seven of them (inhibition > 80%), originating from six plants, namely Cuphea carthagenensis (Lythraceae), Echinodorus grandiflorus (Alismataceae), Mansoa hirsuta (Bignoniaceae), Ouratea semiserrata (Ochnaceae), Ouratea spectabilis and Remijia ferruginea (Rubiaceae). The ethanol extract from O. semiserrata leaves was fractionated over Sephadex LH-20 and RP-HPLC to give three compounds previously reported for the species, along with agathisflavone and epicatechin, here described for the first time in the plant. Epicatechin and lanceoloside A elicited significant inhibition of TNF-α release, indicating that they may account for the effect produced by O. semiserrata crude extract.


Asunto(s)
Extractos Vegetales , Plantas Medicinales , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Bignoniaceae , Brasil , Cromatografía Líquida de Alta Presión , Dextranos , Ochnaceae , Extractos Vegetales/farmacología , Hojas de la Planta , Solventes
9.
Cell Death Differ ; 31(1): 28-39, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38001254

RESUMEN

The ability of cells to mount an interferon response to virus infections depends on intracellular nucleic acid sensing pattern recognition receptors (PRRs). RIG-I is an intracellular PRR that binds short double-stranded viral RNAs to trigger MAVS-dependent signalling. The RIG-I/MAVS signalling complex requires the coordinated activity of multiple kinases and E3 ubiquitin ligases to activate the transcription factors that drive type I and type III interferon production from infected cells. The linear ubiquitin chain assembly complex (LUBAC) regulates the activity of multiple receptor signalling pathways in both ligase-dependent and -independent ways. Here, we show that the three proteins that constitute LUBAC have separate functions in regulating RIG-I signalling. Both HOIP, the E3 ligase capable of generating M1-ubiquitin chains, and LUBAC accessory protein HOIL-1 are required for viral RNA sensing by RIG-I. The third LUBAC component, SHARPIN, is not required for RIG-I signalling. These data cement the role of LUBAC as a positive regulator of RIG-I signalling and as an important component of antiviral innate immune responses.


Asunto(s)
Virus ARN , Ubiquitina-Proteína Ligasas , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Transducción de Señal , Proteína 58 DEAD Box/genética , Virus ARN/metabolismo
10.
PLoS Pathog ; 7(9): e1002247, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21931555

RESUMEN

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-ß (IFN-ß) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-ß expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-ß promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.


Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Virus Vaccinia/genética , Proteínas Virales/genética , Regulación Viral de la Expresión Génica , Genes Reguladores , Células HEK293 , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Evasión Inmune , Inmunidad Innata , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Interferón beta/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Sistemas de Lectura Abierta , Fosforilación , Plásmidos , Unión Proteica/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal , Transcripción Genética , Virus Vaccinia/metabolismo , Virus Vaccinia/fisiología , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Replicación Viral
11.
J Insect Physiol ; 151: 104573, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37838284

RESUMEN

A detailed understanding of how host fitness changes in response to variations in microbe density (an ecological measure of disease tolerance) is an important aim of infection biology. Here, we applied dose-response curves to study Aedes aegypti survival upon exposure to different microbes. We challenged female mosquitoes with Listeria monocytogenes, a model bacterial pathogen, Dengue 4 virus and Zika virus, two medically relevant arboviruses, to understand the distribution of mosquito survival following microbe exposure. By correlating microbe loads and host health, we found that a blood meal promotes disease tolerance in our systemic bacterial infection model and that mosquitoes orally infected with bacteria had an enhanced defensive capacity than insects infected through injection. We also showed that Aedes aegypti displays a higher survival profile following arbovirus infection when compared to bacterial infections. Here, we applied a framework for investigating microbe-induced mosquito mortality and details how the lifespan of Aedes aegypti varies with different inoculum sizes of bacteria and arboviruses.


Asunto(s)
Aedes , Infecciones por Arbovirus , Arbovirus , Virus del Dengue , Infección por el Virus Zika , Virus Zika , Femenino , Animales , Virus del Dengue/fisiología , Mosquitos Vectores/microbiología , Virus Zika/fisiología , Bacterias
12.
Br J Pharmacol ; 180(11): 1460-1481, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36526272

RESUMEN

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.


Asunto(s)
COVID-19 , Sepsis , Humanos , Ratones , Animales , Oseltamivir/efectos adversos , Zanamivir/efectos adversos , Neuraminidasa/metabolismo , Neuraminidasa/farmacología , Neutrófilos , Metaloproteinasa 9 de la Matriz/metabolismo , Especies Reactivas de Oxígeno , Lipopolisacáridos/farmacología , Sepsis/inducido químicamente
13.
J Biol Chem ; 286(23): 20727-35, 2011 Jun 10.
Artículo en Inglés | MEDLINE | ID: mdl-21474453

RESUMEN

The IκB kinase (IKK) complex regulates activation of NF-κB, a critical transcription factor in mediating inflammatory and immune responses. Not surprisingly, therefore, many viruses seek to inhibit NF-κB activation. The vaccinia virus B14 protein contributes to virus virulence by binding to the IKKß subunit of the IKK complex and preventing NF-κB activation in response to pro-inflammatory stimuli. Previous crystallographic studies showed that the B14 protein has a Bcl-2-like fold and forms homodimers in the crystal. However, multi-angle light scattering indicated that B14 is in monomer-dimer equilibrium in solution. This transient self-association suggested that the hydrophobic dimerization interface of B14 might also mediate its interaction with IKKß, and this was investigated by introducing amino acid substitutions on the dimer interface. One mutant (Y35E) was entirely monomeric but still co-immunoprecipitated with IKKß and blocked both NF-κB nuclear translocation and NF-κB-dependent gene expression. Therefore, B14 homodimerization is nonessential for binding and inhibition of IKKß. In contrast, a second monomeric mutant (F130K) neither bound IKKß nor inhibited NF-κB-dependent gene expression, demonstrating that this residue is required for the B14-IKKß interaction. Thus, the dimerization and IKKß-binding interfaces overlap and lie on a surface used for protein-protein interactions in many viral and cellular Bcl-2-like proteins.


Asunto(s)
Núcleo Celular/metabolismo , Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Multimerización de Proteína , Virus Vaccinia/metabolismo , Proteínas Virales/metabolismo , Transporte Activo de Núcleo Celular/genética , Sustitución de Aminoácidos , Núcleo Celular/genética , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Quinasa I-kappa B/genética , Mutación Missense , FN-kappa B/genética , Virus Vaccinia/genética , Proteínas Virales/genética
14.
bioRxiv ; 2022 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-33200130

RESUMEN

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

15.
EBioMedicine ; 77: 103891, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-35220042

RESUMEN

BACKGROUND: Gut microbiota-derived short-chain fatty-acid (SFCA) acetate protects mice against RSV A2 strain infection by increasing interferon-ß production and expression of interferon-stimulated genes (ISGs). However, the role of SFCA in RSV infection using strains isolated from patients is unknown. METHODS: We first used RSV clinical strains isolated from infants hospitalized with RSV bronchiolitis to investigate the effects of in vitro SCFA-acetate treatment of human pulmonary epithelial cells. We next examined whether SCFA-acetate treatment is beneficial in a mouse model of RSV infection using clinical isolates. We sought to investigate the relationship of gut microbiota and fecal acetate with disease severity among infants hospitalized with RSV bronchiolitis, and whether treating their respiratory epithelial cells with SCFA-acetate ex-vivo impacts viral load and ISG expression. We further treated epithelial cells from SARS-CoV-2 infected patients with SCFA-acetate. FINDINGS: In vitro pre-treatment of A549 cells with SCFA-acetate reduced RSV infection with clinical isolates and increased the expression of RIG-I and ISG15. Animals treated with SCFA-acetate intranasally recovered significantly faster, with reduction in the RSV clinical isolates viral load, and increased lung expression of IFNB1 and the RIG-I. Experiments in RIG-I knockout A549 cells demonstrated that the protection relies on RIG-I presence. Gut microbial profile was associated with bronchiolitis severity and with acetate in stool. Increased SCFA-acetate levels were associated with increasing oxygen saturation at admission, and shorter duration of fever. Ex-vivo treatment of patients' respiratory cells with SCFA-acetate reduced RSV load and increased expression of ISGs OAS1 and ISG15, and virus recognition receptors MAVS and RIG-I, but not IFNB1. These SCFA-acetate effects were not found on cells from SARS-CoV-2 infected patients. INTERPRETATION: SCFA-acetate reduces the severity of RSV infection and RSV viral load through modulation of RIG-I expression. FUNDING: FAPERGS (FAPERGS/MS/CNPq/SESRS no. 03/2017 - PPSUS 17/2551-0001380-8 and COVID-19 20/2551-0000258-6); CNPq 312504/2017-9; CAPES) - Finance Code 001.


Asunto(s)
Bronquiolitis , COVID-19 , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Acetatos/metabolismo , Acetatos/farmacología , Animales , Antivirales/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Bronquiolitis/tratamiento farmacológico , Bronquiolitis/metabolismo , Ácidos Grasos Volátiles/metabolismo , Humanos , Lactante , Pulmón/metabolismo , Ratones , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/fisiología , SARS-CoV-2
16.
Elife ; 82019 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-31637998

RESUMEN

Monocyte counts are increased during human tuberculosis (TB) but it has not been determined whether Mycobacterium tuberculosis (Mtb) directly regulates myeloid commitment. We demonstrated that exposure to Mtb directs primary human CD34+ cells to differentiate into monocytes/macrophages. In vitro myeloid conversion did not require type I or type II IFN signaling. In contrast, Mtb enhanced IL-6 responses by CD34+ cell cultures and IL-6R neutralization inhibited myeloid differentiation and decreased mycobacterial growth in vitro. Integrated systems biology analysis of transcriptomic, proteomic and genomic data of large data sets of healthy controls and TB patients established the existence of a myeloid IL-6/IL6R/CEBP gene module associated with disease severity. Furthermore, genetic and functional analysis revealed the IL6/IL6R/CEBP gene module has undergone recent evolutionary selection, including Neanderthal introgression and human pathogen adaptation, connected to systemic monocyte counts. These results suggest Mtb co-opts an evolutionary recent IFN-IL6-CEBP feed-forward loop, increasing myeloid differentiation linked to severe TB in humans.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Interferones/metabolismo , Interleucina-6/metabolismo , Monocitos/metabolismo , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Antígenos CD34 , Proteínas Potenciadoras de Unión a CCAAT/genética , Diferenciación Celular , Proliferación Celular , Citocinas/genética , Citocinas/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Hidrolasas , Interferones/genética , Interleucina-6/genética , Macrófagos/microbiología , Monocitos/microbiología , Mycobacterium tuberculosis/patogenicidad , Células Mieloides/fisiología , Proteómica , Receptores de Interleucina-6 , Índice de Severidad de la Enfermedad , Transcriptoma , Tuberculosis/metabolismo
17.
Viruses ; 9(11)2017 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-29144403

RESUMEN

Herpes simplex virus 1 (HSV-1) has extensive interactions with the host DNA damage response (DDR) machinery that can be either detrimental or beneficial to the virus. Proteins in the homologous recombination pathway are known to be required for efficient replication of the viral genome, while different members of the classical non-homologous end-joining (c-NHEJ) pathway have opposing effects on HSV-1 infection. Here, we have investigated the role of the recently-discovered c-NHEJ component, PAXX (Paralogue of XRCC4 and XLF), which we found to be excluded from the nucleus during HSV-1 infection. We have established that cells lacking PAXX have an intact innate immune response to HSV-1 but show a defect in viral genome replication efficiency. Counterintuitively, PAXX-/- cells were able to produce greater numbers of infectious virions, indicating that PAXX acts to restrict HSV-1 infection in a manner that is different from other c-NHEJ factors.


Asunto(s)
Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Herpes Simple/virología , Herpesvirus Humano 1/fisiología , Animales , Línea Celular , Genes Virales/genética , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/crecimiento & desarrollo , Humanos , Interferones/análisis , Interferones/biosíntesis , Ratones , Proteínas Virales/biosíntesis , Virión/aislamiento & purificación , Replicación Viral
18.
Sci Rep ; 6: 36339, 2016 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-27805018

RESUMEN

Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans.


Asunto(s)
Virus del Dengue/genética , Virus del Dengue/inmunología , Dengue/inmunología , Péptidos/inmunología , Proteínas del Envoltorio Viral/genética , Secuencia de Aminoácidos , Anticuerpos Antivirales/metabolismo , Secuencia de Bases , Secuencia Conservada , Cristalografía por Rayos X , Dengue/virología , Genoma Viral , Humanos , Modelos Moleculares , Mutación , Péptidos/química , Péptidos/genética , Conformación Proteica , Serogrupo , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología
19.
Microbes Infect ; 16(12): 1002-12, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25316508

RESUMEN

Recent years have seen a great advance in knowledge of how a host senses infection. Nucleic acids, as a common denominator to all pathogens, are at the centre of several of the sensing pathways, especially those involved with the recognition of viruses. In this review we discuss the current knowledge on how intracellular DNA is sensed by the mammalian host.


Asunto(s)
ADN Viral/inmunología , Interacciones Huésped-Patógeno , Inmunidad Innata/fisiología , Virosis/inmunología , Virosis/metabolismo , Virus/inmunología , Animales , Autoinmunidad , Humanos , Evasión Inmune , Espacio Intracelular/metabolismo , Transducción de Señal , Vacunas Virales/genética , Vacunas Virales/inmunología , Virosis/prevención & control , Virus/genética
20.
Elife ; 1: e00047, 2012 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-23251783

RESUMEN

Innate immunity is the first immunological defence against pathogens. During virus infection detection of nucleic acids is crucial for the inflammatory response. Here we identify DNA-dependent protein kinase (DNA-PK) as a DNA sensor that activates innate immunity. We show that DNA-PK acts as a pattern recognition receptor, binding cytoplasmic DNA and triggering the transcription of type I interferon (IFN), cytokine and chemokine genes in a manner dependent on IFN regulatory factor 3 (IRF-3), TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING). Both cells and mice lacking DNA-PKcs show attenuated cytokine responses to both DNA and DNA viruses but not to RNA or RNA virus infection. DNA-PK has well-established functions in the DNA repair and V(D)J recombination, hence loss of DNA-PK leads to severe combined immunodeficiency (SCID). However, we now define a novel anti-microbial function for DNA-PK, a finding with implications for host defence, vaccine development and autoimmunity.DOI:http://dx.doi.org/10.7554/eLife.00047.001.


Asunto(s)
ADN Viral/inmunología , Proteína Quinasa Activada por ADN/inmunología , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata , Factor 3 Regulador del Interferón/inmunología , Proteínas Nucleares/inmunología , Animales , Citocinas/biosíntesis , Citocinas/inmunología , Reparación del ADN/inmunología , ADN Viral/metabolismo , Proteína Quinasa Activada por ADN/deficiencia , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Embrión de Mamíferos , Fibroblastos/inmunología , Fibroblastos/virología , Herpesvirus Humano 1/inmunología , Virus de la Influenza A/inmunología , Factor 3 Regulador del Interferón/genética , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Transducción de Señal , Recombinación V(D)J , Virus Vaccinia/inmunología
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