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Chikungunya virus (CHIKV) is a mosquito-borne pathogen responsible for an acute musculoskeletal disease in humans. Replication of the viral RNA genome occurs in specialized membranous replication organelles (ROs) or spherules, which contain the viral replication complex. Initially generated by RNA synthesis-associated plasma membrane deformation, alphavirus ROs are generally rapidly endocytosed to produce type I cytopathic vacuoles (CPV-I), from which nascent RNAs are extruded for cytoplasmic translation. By contrast, CHIKV ROs are poorly internalized, raising the question of their fate and functionality at the late stage of infection. Here, using in situ cryogenic-electron microscopy approaches, we investigate the outcome of CHIKV ROs and associated replication machinery in infected human cells. We evidence the late persistence of CHIKV ROs at the plasma membrane with a crowned protein complex at the spherule neck similar to the recently resolved replication complex. The unexpectedly heterogeneous and large diameter of these compartments suggests a continuous, dynamic growth of these organelles beyond the replication of a single RNA genome. Ultrastructural analysis of surrounding cytoplasmic regions supports that outgrown CHIKV ROs remain dynamically active in viral RNA synthesis and export to the cell cytosol for protein translation. Interestingly, rare ROs with a homogeneous diameter are also marginally internalized in CPV-I near honeycomb-like arrangements of unknown function, which are absent in uninfected controls, thereby suggesting a temporal regulation of this internalization. Altogether, this study sheds new light on the dynamic pattern of CHIKV ROs and associated viral replication at the interface with cell membranes in infected cells.IMPORTANCEThe Chikungunya virus (CHIKV) is a positive-stranded RNA virus that requires specialized membranous replication organelles (ROs) for its genome replication. Our knowledge of this viral cycle stage is still incomplete, notably regarding the fate and functional dynamics of CHIKV ROs in infected cells. Here, we show that CHIKV ROs are maintained at the plasma membrane beyond the first viral cycle, continuing to grow and be dynamically active both in viral RNA replication and in its export to the cell cytosol, where translation occurs in proximity to ROs. This contrasts with the homogeneous diameter of ROs during internalization in cytoplasmic vacuoles, which are often associated with honeycomb-like arrangements of unknown function, suggesting a regulated mechanism. This study sheds new light on the dynamics and fate of CHIKV ROs in human cells and, consequently, on our understanding of the Chikungunya viral cycle.
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Virus Chikungunya , ARN Viral , Replicación Viral , Virus Chikungunya/fisiología , Humanos , ARN Viral/metabolismo , ARN Viral/genética , Fiebre Chikungunya/virología , Compartimentos de Replicación Viral/metabolismo , Orgánulos/virología , Orgánulos/ultraestructura , Orgánulos/metabolismo , Membrana Celular/virología , Membrana Celular/metabolismo , Línea Celular , Microscopía por Crioelectrón , Animales , Genoma ViralRESUMEN
IMPORTANCE: Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new information about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family.
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Dermatosis Nodular Contagiosa , Virus de la Dermatosis Nodular Contagiosa , Proteómica , Proteínas Virales , Animales , Bovinos , Dermatosis Nodular Contagiosa/virología , Virus de la Dermatosis Nodular Contagiosa/metabolismo , Virión/metabolismo , Proteínas Virales/análisis , Proteínas Virales/metabolismo , Proteoma/análisis , Proteoma/metabolismoRESUMEN
In mammalian cells, alphavirus replication complexes are anchored to the plasma membrane. This interaction with lipid bilayers is mediated through the viral methyl/guanylyltransferase nsP1 and reinforced by palmitoylation of cysteine residue(s) in the C-terminal region of this protein. Lipid content of membranes supporting nsP1 anchoring remains poorly studied. Here, we explore the membrane binding capacity of nsP1 with regard to cholesterol. Using the medically important chikungunya virus (CHIKV) as a model, we report that nsP1 cosegregates with cholesterol-rich detergent-resistant membrane microdomains (DRMs), also called lipid rafts. In search for the critical factor for cholesterol partitioning, we identify nsP1 palmitoylated cysteines as major players in this process. In cells infected with CHIKV or transfected with CHIKV trans-replicase plasmids, nsP1, together with the other nonstructural proteins, are detected in DRMs. While the functional importance of CHIKV nsP1 preference for cholesterol-rich membrane domains remains to be determined, we observed that U18666A- and imipramine-induced sequestration of cholesterol in late endosomes redirected nsP1 to these compartments and simultaneously dramatically decreased CHIKV genome replication. A parallel study of Sindbis virus (SINV) revealed that nsP1 from this divergent alphavirus displays a low affinity for cholesterol and only moderately segregates with DRMs. Behaviors of CHIKV and SINV with regard to cholesterol, therefore, match with the previously reported differences in the requirement for nsP1 palmitoylation, which is dispensable for SINV but strictly required for CHIKV replication. Altogether, this study highlights the functional importance of nsP1 segregation with DRMs and provides new insight into the functional role of nsP1 palmitoylated cysteines during alphavirus replication.IMPORTANCE Functional alphavirus replication complexes are anchored to the host cell membranes through the interaction of nsP1 with the lipid bilayers. In this work, we investigate the importance of cholesterol for such an association. We show that nsP1 has affinity for cholesterol-rich membrane microdomains formed at the plasma membrane and identify conserved palmitoylated cysteine(s) in nsP1 as the key determinant for cholesterol affinity. We demonstrate that drug-induced cholesterol sequestration in late endosomes not only redirects nsP1 to this compartment but also dramatically decreases genome replication, suggesting the functional importance of nsP1 targeting to cholesterol-rich plasma membrane microdomains. Finally, we show evidence that nsP1 from chikungunya and Sindbis viruses displays different sensitivity to cholesterol sequestering agents that parallel with their difference in the requirement for nsP1 palmitoylation for replication. This research, therefore, gives new insight into the functional role of palmitoylated cysteines in nsP1 for the assembly of functional alphavirus replication complexes in their mammalian host.
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Virus Chikungunya/metabolismo , Colesterol/metabolismo , Cisteína/metabolismo , Lipoilación/fisiología , Microdominios de Membrana/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Animales , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/virología , Fiebre Chikungunya/virología , Virus Chikungunya/genética , Chlorocebus aethiops , Endosomas/metabolismo , Células HEK293 , Células HeLa , Humanos , Virus Sindbis , Células Vero , Proteínas no Estructurales Virales/genética , Replicación Viral/genéticaRESUMEN
The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.
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Proteínas Bacterianas/metabolismo , Sistemas de Secreción Bacterianos/metabolismo , Coxiella burnetii , Vacuolas/metabolismo , Animales , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Chlorocebus aethiops , Coxiella burnetii/metabolismo , Coxiella burnetii/patogenicidad , Humanos , Lepidópteros/microbiología , Mutación , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , VirulenciaRESUMEN
The replication of viral pathogens relies on their ability to manipulate their host. Several steps of the infectious cycle require the hijacking of cellular membranes. Positive stranded RNA viruses replicating in the cytoplasm of their host reorganize cellular membranes. This leads to the formation of organelles, which host viral replication. The formation of such compartments, which are genuine viral factories, induces morphological modifications of the host cell, which vary depending on the pathogen. However, the molecular mechanisms underlying such a remodeling remain unclear. These mechanisms are subject to intense research since their formation is indispensable to viral multiplication and therefore represent an attractive therapeutic target. In this review, we provide a bird's eye view on the current knowledge of the architecture and virus-host interactions involved in the biogenesis of positive stranded RNA virus replication organelles.
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Orgánulos , Virus ARN Monocatenarios Positivos , Interacciones Microbiota-Huesped , ARN , Replicación Viral/genéticaRESUMEN
HIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promo-ting reverse transcription (RTion) prior to virus release, through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding, restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism.
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Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , VIH-1/fisiología , Factores de Transcripción/metabolismo , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Membrana Celular/virología , ADN Viral/biosíntesis , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Humanos , Transcripción Reversa , Eliminación de Secuencia , Virión/metabolismo , Dedos de Zinc , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
UNLABELLED: Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE: Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.
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Células Dendríticas/virología , Flaviviridae/fisiología , Queratinocitos/virología , Internalización del Virus , Replicación Viral , Aedes/virología , Animales , Autofagia/inmunología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citocinas/biosíntesis , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células Dendríticas/inmunología , Fibroblastos/virología , Flaviviridae/inmunología , Infecciones por Flaviviridae/inmunología , Infecciones por Flaviviridae/virología , Células HEK293 , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Insectos Vectores/virología , Helicasa Inducida por Interferón IFIH1 , Interferón beta/biosíntesis , Interferón beta/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistencia a Mixovirus/biosíntesis , Fagosomas/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos , Receptores Virales/genética , Receptores Virales/metabolismo , Piel/inmunología , Piel/virología , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 3/metabolismo , Receptor Toll-Like 7/inmunología , Ubiquitinas/biosíntesis , Células Vero , Tirosina Quinasa del Receptor AxlRESUMEN
Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated using viruses that easily express detectable marker proteins. Therefore, developing molecular tools, such as reporter-carrying versions of flaviviruses, for studying viral replication and screening antiviral compounds represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods that include the design and validation of infectious clones of Zika, Kunjin, and Dengue viruses harboring different reporter genes for infection, rescue, imaging, and morphology using super-resolution microscopy. It was observed that different flavivirus constructs with identical designs displayed strikingly different genetic stabilities, and corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase the stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies.
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Our study focused on deciphering the role of F-actin and related regulatory factors during SARS-CoV-2 particle production and transmission in human pulmonary cells. Quantitative high-resolution microscopies revealed that the late phases of SARS-CoV-2 infection induce a strong rearrangement of F-actin nanostructures dependent on the viral M, E, and N structural proteins. Intracellular vesicles containing viral components are labeled with Rab7 and Lamp1 and are surrounded by F-actin ring-shaped structures, suggesting their role in viral trafficking toward the cell membrane for virus release. Furthermore, filopodia-like nanostructures were loaded with viruses, potentially facilitating their egress and transmission between lung cells. Gene expression analysis revealed the involvement of alpha-actinins under the regulation of the protein kinase N (PKN). The use of a PKN inhibitor efficiently reduces virus particle production, restoring endoplasmic reticulum and F-actin cellular shape. Our results highlight an important role of F-actin rearrangements during the productive phases of SARS-CoV-2 particles.
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Ras-GTPase-activating SH3 domain-binding-proteins 1 (G3BP1) and 2 (G3BP2) are multifunctional RNA-binding proteins involved in stress granule nucleation, previously identified as essential cofactors of Old World alphaviruses. They are recruited to viral replication complexes formed by the Chikungunya virus (CHIKV), Semliki Forest virus (SFV), and Sindbis virus (SINV) via an interaction with a duplicated FGxF motif conserved in the hypervariable domain (HVD) of virus-encoded nsP3. According to mutagenesis studies, this FGxF duplication is strictly required for G3BP binding and optimal viral growth. Contrasting with this scenario, nsP3 encoded by Mayaro virus (MAYV), an arthritogenic virus grouped with Old World alphaviruses, contains a single canonical FGxF sequence. In light of this unusual feature, we questioned MAYV nsP3/G3BPs relationships. We report that G3BP1 and G3BP2 are both required for MAYV growth in human cells and bind nsP3 protein. In infected cells, they are recruited to nsP3-containing cytosolic foci and active replication complexes. Unexpectedly, deletion of the single FGxF sequence in MAYV nsP3 did not abolish these phenotypes. Using mutagenesis and in silico modeling, we identify an upstream FGAP amino acid sequence as an additional MAYV nsP3/G3BP interaction motif required for optimal viral infectivity. These results, therefore, highlight a non-conventional G3BP binding sequence in MAYV nsP3.
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Virus Chikungunya , Proteínas no Estructurales Virales , ADN Helicasas , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas , Proteínas con Motivos de Reconocimiento de ARN , Replicación ViralRESUMEN
SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.
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Patients with severe COVID-19 show an altered immune response that fails to control the viral spread and suffer from exacerbated inflammatory response, which eventually can lead to death. A major challenge is to develop an effective treatment for COVID-19. NF-κB is a major player in innate immunity and inflammatory process. By a high-throughput screening approach, we identified FDA-approved compounds that inhibit the NF-κB pathway and thus dampen inflammation. Among these, we show that Auranofin prevents post-translational modifications of NF-κB effectors and their recruitment into activating complexes in response to SARS-CoV-2 infection or cytokine stimulation. In addition, we demonstrate that Auranofin counteracts several steps of SARS-CoV-2 infection. First, it inhibits a raft-dependent endocytic pathway involved in SARS-CoV-2 entry into host cells; Second, Auranofin alters the ACE2 mobility at the plasma membrane. Overall, Auranofin should prevent SARS-CoV-2 infection and inflammatory damages, offering new opportunities as a repurposable drug candidate to treat COVID-19.
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BACKGROUND: Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34(+)CD38(low/-) compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34(+)CD38(low/-)CD123(+) phenotype. DESIGN AND METHODS: The percentage of CD34(+)CD38(low/-)CD123(+) cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses. RESULTS: A proportion of CD34(+)CD38(low/-)CD123(+) cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34(+)CD38(low/-)CD123(+) higher than 15% retained significance with an odds ratio of 0.33 (0.1-0.97; P=0.044). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells retained prognostic significance for both parameters after multivariate analysis. CONCLUSIONS: The percentage of CD34(+)CD38(low/-)CD123(+) leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.
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ADP-Ribosil Ciclasa 1/sangre , Antígenos CD34/sangre , Antígenos de Neoplasias/sangre , Crisis Blástica/sangre , Crisis Blástica/diagnóstico , Subunidad alfa del Receptor de Interleucina-3/sangre , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/diagnóstico , Glicoproteínas de Membrana/sangre , Adulto , Anciano , Crisis Blástica/mortalidad , Crisis Blástica/terapia , Femenino , Humanos , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/terapia , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.
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COVID-19/virología , Microscopía de Fuerza Atómica , SARS-CoV-2/ultraestructura , Virión/ultraestructura , Animales , Chlorocebus aethiops , Humanos , SARS-CoV-2/fisiología , Células Vero , Virión/fisiología , Inactivación de VirusRESUMEN
Peptidoglycan (PG) is made of a polymer of disaccharides organized as a three-dimensional mesh-like network connected together by peptidic cross-links. PG is a dynamic structure that is essential for resistance to environmental stressors. Remodeling of PG occurs throughout the bacterial life cycle, particularly during bacterial division and separation into daughter cells. Numerous autolysins with various substrate specificities participate in PG remodeling. Expression of these enzymes must be tightly regulated, as an excess of hydrolytic activity can be detrimental for the bacteria. In non-tuberculous mycobacteria such as Mycobacterium abscessus, the function of PG-modifying enzymes has been poorly investigated. In this study, we characterized the function of the PG amidase, Ami1 from M. abscessus. An ami1 deletion mutant was generated and the phenotypes of the mutant were evaluated with respect to susceptibility to antibiotics and virulence in human macrophages and zebrafish. The capacity of purified Ami1 to hydrolyze muramyl-dipeptide was demonstrated in vitro. In addition, the screening of a 9200 compounds library led to the selection of three compounds inhibiting Ami1 in vitro. We also report the structural characterization of Ami1 which, combined with in silico docking studies, allows us to propose a mode of action for these inhibitors.
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Mycobacterium abscessus/enzimología , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Animales , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Eliminación de Gen , Humanos , Larva/microbiología , Macrófagos/microbiología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/patogenicidad , Mycobacterium abscessus/ultraestructura , N-Acetil Muramoil-L-Alanina Amidasa/antagonistas & inhibidores , Fenotipo , Homología Estructural de Proteína , Células THP-1 , Virulencia , Pez CebraRESUMEN
Infection by rapidly growing Mycobacterium abscessus is increasingly prevalent in cystic fibrosis (CF), a genetic disease caused by a defective CF transmembrane conductance regulator (CFTR). However, the potential link between a dysfunctional CFTR and vulnerability to M. abscessus infection remains unknown. Herein, we exploit a CFTR-depleted zebrafish model, recapitulating CF immuno-pathogenesis, to study the contribution of CFTR in innate immunity against M. abscessus infection. Loss of CFTR increases susceptibility to infection through impaired NADPH oxidase-dependent restriction of intracellular growth and reduced neutrophil chemotaxis, which together compromise granuloma formation and integrity. As a consequence, extracellular multiplication of M. abscessus expands rapidly, inducing abscess formation and causing lethal infections. Because these phenotypes are not observed with other mycobacteria, our findings highlight the crucial and specific role of CFTR in the immune control of M. abscessus by mounting effective oxidative responses.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium abscessus/inmunología , Estrés Oxidativo/inmunología , Proteínas de Pez Cebra/inmunología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Infecciones por Mycobacterium no Tuberculosas/microbiología , Infecciones por Mycobacterium no Tuberculosas/prevención & control , Mycobacterium abscessus/aislamiento & purificación , Especies Reactivas de Oxígeno/inmunología , Pez CebraRESUMEN
Maturation of human immunodeficiency virus type 1 (HIV-1) particles is a key step for viral infectivity. This process can be blocked using maturation inhibitors (MIs) that affect the cleavage of the capsid-spacer peptide 1 (CA-SP1) junction. Here, we investigated the mechanisms underlying the activity of EP-39, a bevirimat (BVM) derivative with better hydrosolubility. To this aim, we selected in vitro EP-39- and BVM-resistant mutants. We found that EP-39-resistant viruses have four mutations within the CA domain (CA-A194T, CA-T200N, CA-V230I, and CA-V230A) and one in the first residue of SP1 (SP1-A1V). We also identified six mutations that confer BVM resistance (CA-A194T, CA-L231F, CA-L231M, SP1-A1V, SP1-S5N and SP1-V7A). To characterize the EP-39 and BVM-resistant mutants, we studied EP-39 effects on mutant virus replication and performed a biochemical analysis with both MIs. We observed common and distinct characteristics, suggesting that, although EP-39 and BVM share the same chemical skeleton, they could interact in a different way with the Gag polyprotein precursor (Pr55Gag). Using an in silico approach, we observed that EP-39 and BVM present different predicted positions on the hexameric crystal structure of the CACTD-SP1 Gag fragment. To clearly understand the relationship between assembly and maturation, we investigated the impact of all identified mutations on virus assembly by expressing Pr55Gag mutants. Finally, using NMR, we have shown that the interaction of EP-39 with a peptide carrying the SP1-A1V mutation (CA-SP1(A1V)-NC) is almost suppressed in comparison with the wild type peptide. These results suggest that EP-39 and BVM could interact differently with the Pr55Gag lattice and that the mutation of the first SP1 residue induces a loss of interaction between Pr55Gag and EP-39.
Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Succinatos/química , Succinatos/farmacología , Triterpenos/química , Triterpenos/farmacología , VIH-1/fisiología , Humanos , Células Jurkat , Simulación del Acoplamiento Molecular , Mutación , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne RNA virus that causes epidemics of debilitating disease in tropical and sub-tropical regions with autochtonous transmission in regions with temperate climate. Currently, there is no licensed vaccine or specific antiviral drug available against CHIKV infection. In this study, we examine the role, in the CHIKV viral cycle, of fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD1), two key lipogenic enzymes required for fatty acid production and early desaturation. We show that both enzymes and their upstream regulator PI3K are required for optimal CHIKV infection. We demonstrate that pharmacologic manipulation of FASN or SCD1 enzymatic activity by non-toxic concentrations of cerulenin or CAY10566 decreases CHIKV genome replication. Interestingly, a similar inhibitory effect was also obtained with Orlistat, an FDA-approved anti-obesity drug that targets FASN activity. These drugs were also effective against Mayaro virus (MAYV), an under-studied arthritogenic Old world Alphavirus endemic in South American countries with potential risk of emergence, urbanization and dispersion to other regions. Altogether, our results identify FASN and SCD1 as conserved druggable cofactors of Alphavirus genome replication and support the broad-spectrum activity of drugs targeting the host fatty acids metabolism.
Asunto(s)
Alphavirus/efectos de los fármacos , Ácido Graso Sintasas/metabolismo , Estearoil-CoA Desaturasa/metabolismo , Replicación Viral/efectos de los fármacos , Alphavirus/genética , Infecciones por Alphavirus/tratamiento farmacológico , Animales , Antivirales/farmacología , Línea Celular , Cerulenina/farmacología , Fiebre Chikungunya/tratamiento farmacológico , Virus Chikungunya/efectos de los fármacos , Virus Chikungunya/genética , Chlorocebus aethiops , Ácido Graso Sintasas/efectos de los fármacos , Genoma Viral , Células HEK293 , Humanos , Orlistat/farmacología , Estearoil-CoA Desaturasa/efectos de los fármacos , Células VeroRESUMEN
A patient with a primary neuroendocrine tumor of the pancreas, presented with severe hypercalcemia. This hypercalcemia of malignancy (HCM) failed to respond to intensive bisphosphonate treatment and needed continuous enhanced diuresis. Only after successful antitumor therapy did the hypercalcemia subside. Hypercalcemia was associated with increased concentrations of plasma PTHrP, calcitonin and 1,25-(OH)(2)D(3). Bone mineral density was markedly increased. We demonstrated the presence of both PTHrP and calcitonin in the tumor at the mRNA and protein level, using RT-PCR, immunohistochemistry and Western blotting. The high levels of plasma PTHrP and the demonstrated predominant renal mechanism in this case of HCM are suspected to be the cause for its refractoriness to bone resorption inhibitors. Our findings furthermore suggest that the tumoral production of calcitonin and PTHrP might have contributed to the increased bone mineral storage of calcium and thus probably attenuated the development of frank hypercalcemia.
Asunto(s)
Hipercalcemia/sangre , Hipercalcemia/etiología , Tumores Neuroendocrinos/sangre , Tumores Neuroendocrinos/complicaciones , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/complicaciones , Secuencia de Bases , Densidad Ósea , Calcitonina/sangre , Calcitonina/genética , Calcitriol/sangre , Diuresis , Humanos , Hipercalcemia/genética , Hipercalcemia/terapia , Masculino , Persona de Mediana Edad , Tumores Neuroendocrinos/genética , Neoplasias Pancreáticas/genética , Proteína Relacionada con la Hormona Paratiroidea/sangre , Proteína Relacionada con la Hormona Paratiroidea/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/genéticaRESUMEN
Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses.