RESUMEN
The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.
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Antivirales , COVID-19 , Humanos , Aciltransferasas/antagonistas & inhibidores , Antivirales/farmacología , SARS-CoV-2 , Linfocitos TRESUMEN
Understanding the factors that influence the airborne survival of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in aerosols is important for identifying routes of transmission and the value of various mitigation strategies for preventing transmission. We present measurements of the stability of SARS-CoV-2 in aerosol droplets (â¼5 to 10 µm equilibrated radius) over timescales spanning 5 s to 20 min using an instrument to probe survival in a small population of droplets (typically 5 to 10) containing â¼1 virus/droplet. Measurements of airborne infectivity change are coupled with a detailed physicochemical analysis of the airborne droplets containing the virus. A decrease in infectivity to â¼10% of the starting value was observable for SARS-CoV-2 over 20 min, with a large proportion of the loss occurring within the first 5 min after aerosolization. The initial rate of infectivity loss was found to correlate with physical transformation of the equilibrating droplet; salts within the droplets crystallize at relative humidities (RHs) below 50%, leading to a near-instant loss of infectivity in 50 to 60% of the virus. However, at 90% RH, the droplet remains homogenous and aqueous, and the viral stability is sustained for the first 2 min, beyond which it decays to only 10% remaining infectious after 10 min. The loss of infectivity at high RH is consistent with an elevation in the pH of the droplets, caused by volatilization of CO2 from bicarbonate buffer within the droplet. Four different variants of SARS-CoV-2 were compared and found to have a similar degree of airborne stability at both high and low RH.
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Partículas y Gotitas de Aerosol , COVID-19 , SARS-CoV-2 , Partículas y Gotitas de Aerosol/química , Partículas y Gotitas de Aerosol/aislamiento & purificación , COVID-19/transmisión , Humanos , Humedad , Concentración de Iones de Hidrógeno , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/patogenicidadRESUMEN
Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.
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COVID-19 , Endosomas , Interacciones Huésped-Patógeno , Neuropilina-1 , SARS-CoV-2 , COVID-19/metabolismo , COVID-19/virología , Sistemas CRISPR-Cas , Endosomas/virología , Eliminación de Gen , Humanos , Nanopartículas , Neuropilina-1/genética , Neuropilina-1/metabolismo , Proteómica , SARS-CoV-2/metabolismo , Nexinas de Clasificación/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.
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COVID-19 , Nanoporos , ARN Guía de Kinetoplastida/química , COVID-19/genética , Genoma Viral/genética , Humanos , Caperuzas de ARN , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/genéticaRESUMEN
The glycan loop of Zika virus (ZIKV) envelope protein (E) contains the glycosylation site and has been well documented to be important for viral pathogenesis and transmission. In the present study, we report that deletions in the E glycan loop, which were recorded in African ZIKV strains previously, have re-emerged in their contemporary Asian lineages. Here, we generated recombinant ZIKV containing specific deletions in the E glycan loop by reverse genetics. Extensive in vitro and in vivo characterization of these deletion mutants demonstrated an attenuated phenotype in an adult A129 mouse model and reduced oral infections in mosquitoes. Surprisingly, these glycan loop deletion mutants exhibited an enhanced neurovirulence phenotype, and resulted in a more severe microcephalic brain in neonatal mouse models. Crystal structures of the ZIKV E protein and a deletion mutant at 2.5 and 2.6 Å, respectively, revealed that deletion of the glycan loop induces encephalitic flavivirus-like conformational alterations, including the appearance of perforations on the surface and a clear change in the topology of the loops. Overall, our results demonstrate that the E glycan loop deletions represent neonatal mouse neurovirulence markers of ZIKV. IMPORTANCE Zika virus (ZIKV) has been identified as a cause of microcephaly and acquired evolutionary mutations since its discovery. Previously deletions in the E glycan loop were recorded in African ZIKV strains, which have re-emerged in the contemporary Asian lineages recently. The glycan loop deletion mutants are not glycosylated, which are attenuated in adult A129 mouse model and reduced oral infections in mosquitoes. More importantly, the glycan loop deletion mutants induce an encephalitic flavivirus-like conformational alteration in the E homodimer, resulting in a significant enhancement of neonatal mouse neurovirulence. This study underscores the critical role of glycan loop deletion mutants in ZIKV pathogenesis, highlighting a need for global virological surveillance for such ZIKV variants.
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Proteínas del Envoltorio Viral , Infección por el Virus Zika , Virus Zika , Animales , Ratones , Modelos Animales de Enfermedad , Polisacáridos/química , Proteínas del Envoltorio Viral/genética , Virulencia , Replicación Viral/genética , Virus Zika/genética , Virus Zika/patogenicidad , Infección por el Virus Zika/virologíaRESUMEN
Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike's polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.
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COVID-19/virología , Furina/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , SARS-CoV-2/fisiología , Serina Endopeptidasas/metabolismo , Línea Celular , Endosomas/metabolismo , Furina/genética , Expresión Génica , Humanos , Evasión Inmune , Interferones/metabolismo , Lisosomas/enzimología , Coactivadores de Receptor Nuclear/genética , Isoformas de Proteínas , Proteolisis , Serina Endopeptidasas/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Pseudotipado Viral , Internalización del VirusRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a broad range of clinical responses including prominent microvascular damage. The capacity of SARS-CoV-2 to infect vascular cells is still debated. Additionally, the SARS-CoV-2 Spike (S) protein may act as a ligand to induce non-infective cellular stress. We tested this hypothesis in pericytes (PCs), which are reportedly reduced in the heart of patients with severe coronavirus disease-2019 (COVID-19). Here we newly show that the in vitro exposure of primary human cardiac PCs to the SARS-CoV-2 wildtype strain or the α and δ variants caused rare infection events. Exposure to the recombinant S protein alone elicited signalling and functional alterations, including: (1) increased migration, (2) reduced ability to support endothelial cell (EC) network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm, and (4) production of pro-apoptotic factors causing EC death. Next, adopting a blocking strategy against the S protein receptors angiotensin-converting enzyme 2 (ACE2) and CD147, we discovered that the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in PCs. The neutralisation of CD147, either using a blocking antibody or mRNA silencing, reduced ERK1/2 activation, and rescued PC function in the presence of the S protein. Immunoreactive S protein was detected in the peripheral blood of infected patients. In conclusion, our findings suggest that the S protein may prompt PC dysfunction, potentially contributing to microvascular injury. This mechanism may have clinical and therapeutic implications.
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Enzima Convertidora de Angiotensina 2/metabolismo , Basigina/metabolismo , Miocardio/enzimología , Pericitos/enzimología , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , COVID-19/sangre , Células CACO-2 , Muerte Celular , Niño , Preescolar , Citocinas/metabolismo , Femenino , Interacciones Huésped-Patógeno , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Miocardio/citología , Pericitos/virología , Cultivo Primario de Células , Adulto JovenRESUMEN
We investigate binding of linoleate and other potential ligands to the recently discovered fatty acid binding site in the SARS-CoV-2 spike protein, using docking and molecular dynamics simulations. Simulations suggest that linoleate and dexamethasone stabilize the locked spike conformation, thus reducing the opportunity for ACE2 interaction. In contrast, cholesterol may expose the receptor-binding domain by destabilizing the closed structure, preferentially binding to a different site in the hinge region of the open structure. We docked a library of FDA-approved drugs to the fatty acid site using an approach that reproduces the structure of the linoleate complex. Docking identifies steroids (including dexamethasone and vitaminâ D); retinoids (some known to be active in vitro, and vitaminâ A); and vitaminâ K as potential ligands that may stabilize the closed conformation. The SARS-CoV-2 spike fatty acid site may bind a diverse array of ligands, including dietary components, and therefore provides a promising target for therapeutics or prophylaxis.
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Simulación de Dinámica Molecular , Retinoides/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Esteroides/metabolismo , Vitaminas/metabolismo , Sitios de Unión , COVID-19/patología , COVID-19/virología , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Estructura Cuaternaria de Proteína , Retinoides/química , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Esteroides/química , Vitaminas/químicaAsunto(s)
Microbiología del Aire , Aerosoles y Gotitas Respiratorias , Infecciones del Sistema Respiratorio , Virosis , Tos , Humanos , Concentración de Iones de Hidrógeno , Aerosoles y Gotitas Respiratorias/virología , Infecciones del Sistema Respiratorio/transmisión , Infecciones del Sistema Respiratorio/virología , Virosis/transmisiónRESUMEN
Subunit vaccines use delivery platforms to present minimal antigenic components for immunization. The benefits of such systems include multivalency, self-adjuvanting properties, and more specific immune responses. Previously, the design, synthesis, and characterization of self-assembling peptide cages (SAGEs) have been reported. In these, de novo peptides are combined to make hubs that assemble into nanoparticles when mixed in aqueous solution. Here it is shown that SAGEs are nontoxic particles with potential as accessible synthetic peptide scaffolds for the delivery of immunogenic components. To this end, SAGEs functionalized with the model antigenic peptides tetanus toxoid632-651 and ovalbumin323-339 drive antigen-specific responses both in vitro and in vivo, eliciting both CD4+ T cell and B cell responses. Additionally, SAGEs functionalized with the antigenic peptide hemagglutinin518-526 from the influenza virus are also able to drive a CD8+ T cell response in vivo. This work demonstrates the potential of SAGEs to act as a modular scaffold for antigen delivery, capable of inducing and boosting specific and tailored immune responses.
RESUMEN
BACKGROUND: Aedes aegypti is a vector for the (re-)emerging human pathogens dengue, chikungunya, yellow fever and Zika viruses. Almost half of the Ae. aegypti genome is comprised of transposable elements (TEs). Transposons have been linked to diverse cellular processes, including the establishment of viral persistence in insects, an essential step in the transmission of vector-borne viruses. However, up until now it has not been possible to study the overall proteome derived from an organism's mobile genetic elements, partly due to the highly divergent nature of TEs. Furthermore, as for many non-model organisms, incomplete genome annotation has hampered proteomic studies on Ae. aegypti. RESULTS: We analysed the Ae. aegypti proteome using our new proteomics informed by transcriptomics (PIT) technique, which bypasses the need for genome annotation by identifying proteins through matched transcriptomic (rather than genomic) data. Our data vastly increase the number of experimentally confirmed Ae. aegypti proteins. The PIT analysis also identified hotspots of incomplete genome annotation, and showed that poor sequence and assembly quality do not explain all annotation gaps. Finally, in a proof-of-principle study, we developed criteria for the characterisation of proteomically active TEs. Protein expression did not correlate with a TE's genomic abundance at different levels of classification. Most notably, long terminal repeat (LTR) retrotransposons were markedly enriched compared to other elements. PIT was superior to 'conventional' proteomic approaches in both our transposon and genome annotation analyses. CONCLUSIONS: We present the first proteomic characterisation of an organism's repertoire of mobile genetic elements, which will open new avenues of research into the function of transposon proteins in health and disease. Furthermore, our study provides a proof-of-concept that PIT can be used to evaluate a genome's annotation to guide annotation efforts which has the potential to improve the efficiency of annotation projects in non-model organisms. PIT therefore represents a valuable new tool to study the biology of the important vector species Ae. aegypti, including its role in transmitting emerging viruses of global public health concern.
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Aedes/metabolismo , Elementos Transponibles de ADN/genética , Genoma , Proteoma/análisis , Proteómica/métodos , Aedes/genética , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Mapeo Contig , Proteínas de Insectos/análisis , Proteínas de Insectos/aislamiento & purificación , ARN/aislamiento & purificación , ARN/metabolismo , Análisis de Secuencia de ARN , Espectrometría de Masas en TándemRESUMEN
Smoothness is a hallmark of healthy movement. Past research indicates that smoothness may be a side product of a control strategy that minimizes error. However, this is not the only reason for smooth movements. Our musculoskeletal system itself contributes to movement smoothness: the mechanical impedance (inertia, damping, and stiffness) of our limbs and joints resists sudden change, resulting in a natural smoothing effect. How the biomechanics and neural control interact to result in an observed level of smoothness is not clear. The purpose of this study is to 1) characterize the smoothness of wrist rotations, 2) compare it with the smoothness of planar shoulder-elbow (reaching) movements, and 3) determine the cause of observed differences in smoothness. Ten healthy subjects performed wrist and reaching movements involving different targets, directions, and speeds. We found wrist movements to be significantly less smooth than reaching movements and to vary in smoothness with movement direction. To identify the causes underlying these observations, we tested a number of hypotheses involving differences in bandwidth, signal-dependent noise, speed, impedance anisotropy, and movement duration. Our simulations revealed that proximal-distal differences in smoothness reflect proximal-distal differences in biomechanics: the greater impedance of the shoulder-elbow filters neural noise more than the wrist. In contrast, differences in signal-dependent noise and speed were not sufficiently large to recreate the observed differences in smoothness. We also found that the variation in wrist movement smoothness with direction appear to be caused by, or at least correlated with, differences in movement duration, not impedance anisotropy.NEW & NOTEWORTHY This article presents the first thorough characterization of the smoothness of wrist rotations (flexion-extension and radial-ulnar deviation) and comparison with the smoothness of reaching (shoulder-elbow) movements. We found wrist rotations to be significantly less smooth than reaching movements and determined that this difference reflects proximal-distal differences in biomechanics: the greater impedance (inertia, damping, stiffness) of the shoulder-elbow filters noise in the command signal more than the impedance of the wrist.
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Movimiento/fisiología , Rango del Movimiento Articular/fisiología , Rotación , Adolescente , Adulto , Análisis de Varianza , Brazo/fisiología , Fenómenos Biomecánicos/fisiología , Simulación por Computador , Retroalimentación Sensorial , Femenino , Humanos , Articulaciones/inervación , Masculino , Modelos Biológicos , Dinámicas no Lineales , Torque , Muñeca/inervación , Adulto JovenRESUMEN
Feline infectious peritonitis (FIP) is a fatal disease of cats, and a sequela of systemic feline coronavirus (FCoV) infection. Mutations in the viral spike (S) gene have been associated with FCoVs found in tissues from cats with FIP, but not FCoVs found in faeces from healthy cats, and are implicated in monocyte/macrophage tropism and systemic spread. This study was designed to determine whether S gene mutation analysis can reliably diagnose FIP. Cats were categorised as with FIP (n = 57) or without FIP (n = 45) based on gross post-mortem and histopathological examination including immunohistochemistry for FCoV antigen. RNA was purified from available tissue, fluid and faeces. Reverse-transcriptase quantitative-PCR (RT-qPCR) was performed on all samples using FCoV-specific primers, followed by sequencing of a section of the S gene on RT-qPCR positive samples. Samples were available from a total of 102 cats. Tissue, fluid, and faecal samples from cats with FIP were more likely to be FCoV RT-qPCR-positive (90.4, 78.4 and 64.6% respectively) than those from cats without FIP (7.8, 2.1 and 20% respectively). Identification of S gene mutated FCoVs as an additional step to the detection of FCoV alone, only moderately increased specificity for tissue samples (from 92.6 to 94.6%) but specificity was unchanged for fluid samples (97.9%) for FIP diagnosis; however, sensitivity was markedly decreased for tissue (from 89.8 to 80.9%) and fluid samples (from 78.4 to 60%) for FIP diagnosis. These findings demonstrate that S gene mutation analysis in FCoVs does not substantially improve the ability to diagnose FIP as compared to detection of FCoV alone.
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Coronavirus Felino/genética , Peritonitis Infecciosa Felina/diagnóstico , Glicoproteína de la Espiga del Coronavirus/genética , Animales , Antígenos Virales/genética , Gatos , Heces/virología , Peritonitis Infecciosa Felina/virología , Genes Virales/genética , Mutación/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Análisis de Secuencia de ADN/veterinariaRESUMEN
Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.
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Células Dendríticas/virología , Virus del Dengue , Proteínas del Envoltorio Viral/metabolismo , Citoesqueleto de Actina/metabolismo , Evolución Biológica , Línea Celular , Humanos , Inmunidad Innata/inmunología , Fusión de Membrana/fisiologíaRESUMEN
The four serotypes of dengue virus (DENV-1 to -4) cause the most important arthropod-borne viral disease of humans. DENV non-structural protein 5 (NS5) contains enzymatic activities required for capping and replication of the viral RNA genome that occurs in the host cytoplasm. However, previous studies have shown that DENV-2 NS5 accumulates in the nucleus during infection. In this study, we examined the nuclear localization of NS5 for all four DENV serotypes. We demonstrate for the first time that there are serotypic differences in NS5 nuclear localization. Whereas the DENV-2 and -3 proteins accumulate in the nucleus, DENV-1 and -4 NS5 are predominantly if not exclusively localized to the cytoplasm. Comparative studies on the DENV-2 and -4 NS5 proteins revealed that the difference in DENV-4 NS5 nuclear localization was not due to rapid nuclear export but rather the lack of a functional nuclear localization sequence. Interaction studies using DENV-2 and -4 NS5 and human importin-α isoforms failed to identify an interaction that supported the differential nuclear localization of NS5. siRNA knockdown of the human importin-α isoform KPNA2, corresponding to the murine importin-α isoform previously shown to bind to DENV-2 NS5, did not substantially affect DENV-2 NS5 nuclear localization, whereas knockdown of importin-ß did. The serotypic differences in NS5 nuclear localization did not correlate with differences in IL-8 gene expression. The results show that NS5 nuclear localization is not strictly required for virus replication but is more likely to have an auxiliary function in the life cycle of specific DENV serotypes.
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Núcleo Celular/metabolismo , Virus del Dengue/fisiología , Proteínas Estructurales Virales/metabolismo , Línea Celular , Movimiento Celular , HumanosRESUMEN
The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263-271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1-4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265-900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272-900) reported previously. This new orthorhombic crystal form (space group P21212) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed "preinitiation" conformation similar to the one that was captured previously in space group C2221 with one molecule per asymmetric unit. The structure reveals that residues 269-271 interact with the RdRp domain and suggests that residues 263-268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.
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Virus del Dengue/fisiología , Metiltransferasas/química , ARN Polimerasa Dependiente del ARN/química , Proteínas no Estructurales Virales/química , Replicación Viral/fisiología , Estabilidad de Enzimas/fisiología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutación , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Coronaviruses pose a permanent risk of outbreaks, with three highly pathogenic species and strains (SARS-CoV, MERS-CoV, SARS-CoV-2) having emerged in the last twenty years. Limited antiviral therapies are currently available and their efficacy in randomized clinical trials enrolling SARS-CoV-2 patients has not been consistent, highlighting the need for more potent treatments. We previously showed that cobicistat, a clinically approved inhibitor of Cytochrome P450-3A (CYP3A), has direct antiviral activity against early circulating SARS-CoV-2 strains in vitro and in Syrian hamsters. Cobicistat is a derivative of ritonavir, which is co-administered as pharmacoenhancer with the SARS-CoV-2 protease inhibitor nirmatrelvir, to inhibit its metabolization by CPY3A and preserve its antiviral efficacy. Here, we used automated image analysis for a screening and parallel comparison of the anti-coronavirus effects of cobicistat and ritonavir. Our data show that both drugs display antiviral activity at low micromolar concentrations against multiple SARS-CoV-2 variants in vitro, including epidemiologically relevant Omicron subvariants. Despite their close structural similarity, we found that cobicistat is more potent than ritonavir, as shown by significantly lower EC50 values in monotherapy and higher levels of viral suppression when used in combination with nirmatrelvir. Finally, we show that the antiviral activity of both cobicistat and ritonavir is maintained against other human coronaviruses, including HCoV-229E and the highly pathogenic MERS-CoV. Overall, our results demonstrate that cobicistat has more potent anti-coronavirus activity than ritonavir and suggest that dose adjustments could pave the way to the use of both drugs as broad-spectrum antivirals against highly pathogenic human coronaviruses.
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Infecciones por Coronavirus , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , Antivirales/uso terapéutico , Ritonavir/farmacología , Inhibidores del Citocromo P-450 CYP3A/farmacología , Inhibidores del Citocromo P-450 CYP3A/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Cobicistat/uso terapéuticoRESUMEN
Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , SARS-CoV-2/ultraestructura , Humanos , COVID-19/virología , Células Vero , Chlorocebus aethiops , Animales , Microscopía Electrónica de Transmisión/métodos , Replicación Viral , Microscopía Electrónica/métodosRESUMEN
An improved understanding of the underlying physicochemical properties of respiratory aerosol that influence viral infectivity may open new avenues to mitigate the transmission of respiratory diseases such as COVID-19. Previous studies have shown that an increase in the pH of respiratory aerosols following generation due to changes in the gas-particle partitioning of pH buffering bicarbonate ions and carbon dioxide is a significant factor in reducing SARS-CoV-2 infectivity. We show here that a significant increase in SARS-CoV-2 aerostability results from a moderate increase in the atmospheric carbon dioxide concentration (e.g. 800 ppm), an effect that is more marked than that observed for changes in relative humidity. We model the likelihood of COVID-19 transmission on the ambient concentration of CO2, concluding that even this moderate increase in CO2 concentration results in a significant increase in overall risk. These observations confirm the critical importance of ventilation and maintaining low CO2 concentrations in indoor environments for mitigating disease transmission. Moreover, the correlation of increased CO2 concentration with viral aerostability need to be better understood when considering the consequences of increases in ambient CO2 levels in our atmosphere.
Asunto(s)
COVID-19 , Dióxido de Carbono , SARS-CoV-2 , Dióxido de Carbono/metabolismo , Dióxido de Carbono/análisis , COVID-19/transmisión , COVID-19/virología , Humanos , Concentración de Iones de Hidrógeno , Aerosoles , Humedad , Ventilación , Aerosoles y Gotitas Respiratorias/metabolismo , Aerosoles y Gotitas Respiratorias/virología , Atmósfera/químicaRESUMEN
The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.