RESUMEN
The Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging pathogen of the Orthonairovirus genus that can cause severe and often lethal hemorrhagic diseases in humans. CCHFV has a broad tropism and can infect a variety of species and tissues. Here, by using gene silencing, blocking antibodies or soluble receptor fragments, we identify the low-density lipoprotein receptor (LDL-R) as a CCHFV entry factor. The LDL-R facilitates binding of CCHFV particles but does not allow entry of Hazara virus (HAZV), another member of the genus. In addition, we show that apolipoprotein E (apoE), an exchangeable protein that mediates LDL/LDL-R interaction, is incorporated on CCHFV particles, though not on HAZV particles, and enhances their specific infectivity by promoting an LDL-R dependent entry. Finally, we show that molecules that decrease LDL-R from the surface of target cells could inhibit CCHFV infection. Our study highlights that CCHFV takes advantage of a lipoprotein receptor and recruits its natural ligand to promote entry into cells.
Asunto(s)
Apolipoproteínas E , Virus de la Fiebre Hemorrágica de Crimea-Congo , Receptores de LDL , Internalización del Virus , Humanos , Receptores de LDL/metabolismo , Apolipoproteínas E/metabolismo , Apolipoproteínas E/genética , Virus de la Fiebre Hemorrágica de Crimea-Congo/metabolismo , Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Animales , Células HEK293 , Chlorocebus aethiops , Fiebre Hemorrágica de Crimea/virología , Fiebre Hemorrágica de Crimea/metabolismo , Virión/metabolismo , Células VeroRESUMEN
We developed new iminosugar-based glycosidase inhibitors against SARS-CoV-2. Known drugs (miglustat, migalastat, miglitol, and swainsonine) were chosen as lead compounds to develop three classes of glycosidase inhibitors (α-glucosidase, α-galactosidase, and mannosidase). Molecular modelling of the lead compounds, synthesis of the compounds with the highest docking scores, enzyme inhibition tests, and in vitro antiviral assays afforded rationally designed inhibitors. Two highly active α-glucosidase inhibitors were discovered, where one of them is the most potent iminosugar-based anti-SARS-CoV-2 agent to date (EC90 = 1.94 µM in A549-ACE2 cells against Omicron BA.1 strain). However, galactosidase inhibitors did not exhibit antiviral activity, whereas mannosidase inhibitors were both active and cytotoxic. As our iminosugar-based drug candidates act by a host-directed mechanism, they should be more resilient to drug resistance. Moreover, this strategy could be extended to identify potential drug candidates for other viral infections.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Modelos Moleculares , Manosidasas , Antivirales/farmacología , Simulación del Acoplamiento MolecularRESUMEN
Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.
Asunto(s)
Aedes , Infecciones por Alphavirus , Alphavirus , Arbovirus , Virus Chikungunya , Animales , Ratones , Humanos , Aedes/genética , Alphavirus/genética , Virus Chikungunya/genética , Mosquitos Vectores/genética , Glicoproteínas , Inmunoglobulinas , Proteínas de la MembranaRESUMEN
Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identify DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX is sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricts infection. DAXX restricts an early, post-entry step of the SARS-CoV-2 life cycle. DAXX-mediated restriction of SARS-CoV-2 is independent of the SUMOylation pathway but dependent on its D/E domain, also necessary for its protein-folding activity. SARS-CoV-2 infection triggers the re-localization of DAXX to cytoplasmic sites and promotes its degradation. Mechanistically, this process is mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.
Asunto(s)
COVID-19 , SARS-CoV-2 , Sistemas CRISPR-Cas , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Humanos , Interferones/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
Background: Self-amplifying mRNA is the next-generation vaccine platform with the potential advantages in efficacy and speed of development against infectious diseases and cancer. The main aim was to present optimized and rapid methods for Semliki Forest virus (SFV)-PD self-amplifying mRNA (SAM) preparation, its packaging, and titer determination. These protocols are provided for producing and harvesting the high yields of virus replicon particle (VRP)-packaged SAM for vaccine studies. Methods: pSFV-PD-EGFP plasmid was linearized and subjected to in vitro transcription. Different concentrations of SFV-PD SAM were first transfected into human embryonic kidney 293 cells (HEK-293) and baby hamster kidney cell line 21 (BHK-21) cell lines, and EGFP expression at different time points was evaluated by fluorescent microscopy. Replicon particle packaging was achieved by co-transfection of SFV-PD SAM and pSFV-Helper2 RNA into BHK-21 cells. The VRPs were concentrated using ultrafiltration with 100 kDa cut-off. The titers of replicon particles were determined by reverse transcription quantitative real-time PCR (RT-qPCR). Results: In vitro transcribed SAM encoding EGFP was successfully transfected and expressed in HEK-293 and BHK-21 cell lines. Higher levels of EGFP expression was observed in BHK-21 compared to HEK-293 cells showing more stable protein overexpression and VRP packaging. Using ultrafiltration, the high yields of purified SFV-PD-EGFP particles were rapidly obtained with only minor loss of replicon particles. Accurate and rapid titer determination of replication-deficient particles was achieved by RT-qPCR. Conclusion: Using optimized methods for SAM transfection, VRP packaging, and concentration, high yields of SFV-PD VRPs could be produced and purified. The RT-qPCR demonstrated to be an accurate and rapid method for titer determination of replication deficient VRPs.
Asunto(s)
Vectores Genéticos , Virus de los Bosques Semliki , Animales , Cricetinae , Células HEK293 , Humanos , ARN Mensajero , Transfección , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
The main aims of the present study were to design a fusion protein of Leishmania major stress-inducible protein 1 (LmSTI1) and Phlebotomus papatasi SP15 (PpSP15), and to express it in the form of alphavirus packaged Self-amplifying mRNA (SAM). Two combinations, PpSP15-LmSTI1 and LmSTI1-PpSP15 fusion forms, were analyzed for folding and minimum free energies of the mRNA. Conformational studies on 3D modeled fusion and native forms were performed, and the Root-Mean-Square-distance (RMSD) of the Cα atomic coordinates were calculated. Antigenicity and stability were predicted using bioinformatics tools. The coding sequences of PpSP15-LmSTI1 fusion, PpSP15, and LmSTI1 were cloned into an alphavirus-based vector and used to produce the SAM constructs. All the subcloned constructs were then subjected to packaging in the form of viral replicon particles (VRPs),and were evaluated for their ability to infect BHK-21 cells and express the recombinant fusion proteins. The in-silico analysis indicated that the PpSP15-LmSTI1 combination could be a promising candidate based on lower folding ΔG of mRNA, higher protein antigenicity and lower instability indexes, and less conformational changes compared to the native proteins and the LmSTI1-PpSP15 fusion form. Packaged SAM encoding fusion and native antigens are used for infection of mammalian cells and for recombinant protein expression. This is the first study on in silico designing and successful packaging of an alphavirus-derived SAM in the form of the VRPs to target leishmaniasis.
Asunto(s)
Alphavirus , Leishmania major , Leishmaniasis Cutánea , Phlebotomus , Vacunas , Alphavirus/genética , Animales , Leishmania major/genética , Mamíferos , Phlebotomus/genética , ARN Mensajero/genética , Proteínas RecombinantesRESUMEN
Single-nucleotide polymorphism in APOBEC3C (resulting in a serine to isoleucine in position 188) is present in approximately 10% of African populations and greatly enhances restriction against human immunodeficiency virus-1 and simian immunodeficiency virus by improving dimerization and DNA processivity of the enzyme. In this study, we demonstrated in culture and in infected patients that hepatitis B virus (HBV) could be edited by APOBEC3CS188I. Using next-generation sequencing, we demonstrated that APOBEC3CS188I led to enhanced editing activity in 5'TpCpAâ5'TpTpA context. This constitutes a new hallmark of this enzyme, which could be used to determine its impact on HBV or nuclear DNA.
Asunto(s)
Citidina Desaminasa , Genoma Viral , Virus de la Hepatitis B , Citidina Desaminasa/genética , Hepatitis B/genética , Virus de la Hepatitis B/genética , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
SARS-CoV-2 infection results in impaired interferon response in patients with severe COVID-19. However, how SARS-CoV-2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS-CoV-2-infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus-derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3'UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 can potentially employ a virus-derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon-mediated immune response.
Asunto(s)
COVID-19 , MicroARNs , ARN Viral/genética , SARS-CoV-2/genética , Regiones no Traducidas 3' , COVID-19/inmunología , Humanos , Inmunidad , MicroARNs/genéticaRESUMEN
Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.
Asunto(s)
Cápside/química , Mutación/efectos de los fármacos , Rhinovirus/fisiología , Desencapsidación Viral/fisiología , Antivirales/farmacología , Cápside/efectos de los fármacos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Endosomas/química , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Molécula 1 de Adhesión Intercelular/metabolismo , Conformación Proteica , Rhinovirus/química , Rhinovirus/efectos de los fármacos , Rhinovirus/genética , Virión/química , Virión/genética , Virión/metabolismo , Internalización del Virus/efectos de los fármacos , Desencapsidación Viral/efectos de los fármacos , Desencapsidación Viral/genéticaRESUMEN
Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
Asunto(s)
Pirazinas/química , Virus ARN/genética , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Recombinación Genética , Ribonucleótidos/química , Animales , Antivirales , Catálisis , Células Cultivadas , Técnicas Genéticas , Genoma , Genoma Viral , Recombinación Homóloga , Humanos , Cinética , Ratones , Ratones Transgénicos , Simulación de Dinámica Molecular , Mutagénesis , Nucleótidos/genética , Conformación Proteica , ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , RNA-Seq , Transgenes , VirulenciaRESUMEN
Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Reposicionamiento de Medicamentos , Lipidosis/inducido químicamente , Fosfolípidos/metabolismo , SARS-CoV-2/efectos de los fármacos , Células A549 , Animales , Antivirales/química , Antivirales/uso terapéutico , Antivirales/toxicidad , COVID-19/virología , Cationes , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , SARS-CoV-2/fisiología , Tensoactivos/química , Tensoactivos/farmacología , Tensoactivos/toxicidad , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
New vaccine platforms are crucial to address complex parasitic infections such as cutaneous leishmaniasis. Self-amplifying mRNA (SAM) based vaccines represent the next generation nucleic acid-based platform. In the present study, we compared the expression levels of PpSP15-LmSTI1 fusion gene in BHK-21 cells following transfection with Semliki Forest virus (SFV)-derived SAM, SFV-derived plasmid DNA (pSFV-PD) and conventional plasmid DNA (pcDNA3.1+). PpSP15-LmSTI1 fusion gene expression levels were evaluated at different time points, using quantitative Real-time PCR. All data were validated and normalized by two internal control genes. According to the results, mean values of relative expression were significantly higher for SFV-PD SAM/fusion than pcDNA/fusion and pSFV-PD/fusion at all concentrations and time points. Our results showed that higher levels of PpSp15-LmSTI1 antigen expression could be achieved using a SAM vector than pcDNA and pSFV-PD, making it a valuable and efficient alternative to conventional plasmid DNA-based vaccines against leishmaniasis.
Asunto(s)
Alphavirus , Vacunas de ADN , Alphavirus/genética , Expresión Génica , ARN Mensajero/genética , Transfección , Vacunas de ADN/genéticaRESUMEN
Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.
Asunto(s)
Antivirales/administración & dosificación , Virus Defectuosos/genética , Mosquitos Vectores/efectos de los fármacos , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/genética , Aedes/efectos de los fármacos , Aedes/virología , Animales , Chlorocebus aethiops , Biología Computacional , Evolución Molecular Dirigida , Modelos Animales de Enfermedad , Femenino , Aptitud Genética , Genoma Viral/genética , Células HEK293 , Humanos , Ratones , Control de Mosquitos/métodos , Mosquitos Vectores/virología , Sistemas de Lectura Abierta/genética , ARN Viral/genética , Células Vero , Infección por el Virus Zika/transmisión , Infección por el Virus Zika/virologíaRESUMEN
Repurposing drugs as treatments for COVID-19 has drawn much attention. A common strategy has been to screen for established drugs, typically developed for other indications, that are antiviral in cells or organisms. Intriguingly, most of the drugs that have emerged from these campaigns, though diverse in structure, share a common physical property: cationic amphiphilicity. Provoked by the similarity of these repurposed drugs to those inducing phospholipidosis, a well-known drug side effect, we investigated phospholipidosis as a mechanism for antiviral activity. We tested 23 cationic amphiphilic drugs-including those from phenotypic screens and others that we ourselves had found-for induction of phospholipidosis in cell culture. We found that most of the repurposed drugs, which included hydroxychloroquine, azithromycin, amiodarone, and four others that have already progressed to clinical trials, induced phospholipidosis in the same concentration range as their antiviral activity; indeed, there was a strong monotonic correlation between antiviral efficacy and the magnitude of the phospholipidosis. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the gross physical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a confound in early drug discovery. Understanding its role in infection, and detecting its effects rapidly, will allow the community to better distinguish between drugs and lead compounds that more directly impact COVID-19 from the large proportion of molecules that manifest this confounding effect, saving much time, effort and cost.
RESUMEN
Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.
Asunto(s)
Aedes/virología , Antivirales/farmacología , Fiebre Chikungunya/transmisión , Virus Chikungunya/genética , Virus Defectuosos/genética , Genoma Viral , Replicación Viral , Animales , Fiebre Chikungunya/inmunología , Fiebre Chikungunya/virología , Virus Chikungunya/crecimiento & desarrollo , Virus Chikungunya/aislamiento & purificación , Humanos , Mosquitos Vectores/virologíaRESUMEN
The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
Asunto(s)
COVID-19/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Interacciones Microbiota-Huesped , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Mapas de Interacción de Proteínas , SARS-CoV-2/metabolismo , Síndrome Respiratorio Agudo Grave/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Secuencia Conservada , Proteínas de la Nucleocápside de Coronavirus/genética , Microscopía por Crioelectrón , Humanos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Conformación ProteicaRESUMEN
The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected millions and killed hundreds of thousands of people worldwide, highlighting an urgent need to develop antiviral therapies. Here we present a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in Vero E6 cells, revealing dramatic rewiring of phosphorylation on host and viral proteins. SARS-CoV-2 infection promoted casein kinase II (CK2) and p38 MAPK activation, production of diverse cytokines, and shutdown of mitotic kinases, resulting in cell cycle arrest. Infection also stimulated a marked induction of CK2-containing filopodial protrusions possessing budding viral particles. Eighty-seven drugs and compounds were identified by mapping global phosphorylation profiles to dysregulated kinases and pathways. We found pharmacologic inhibition of the p38, CK2, CDK, AXL, and PIKFYVE kinases to possess antiviral efficacy, representing potential COVID-19 therapies.
Asunto(s)
Betacoronavirus/metabolismo , Infecciones por Coronavirus/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Neumonía Viral/metabolismo , Proteómica/métodos , Células A549 , Enzima Convertidora de Angiotensina 2 , Animales , Antivirales/farmacología , COVID-19 , Células CACO-2 , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Chlorocebus aethiops , Infecciones por Coronavirus/virología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Pandemias , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Fosforilación , Neumonía Viral/virología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
Asunto(s)
Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/metabolismo , Reposicionamiento de Medicamentos , Terapia Molecular Dirigida , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/metabolismo , Mapas de Interacción de Proteínas , Proteínas Virales/metabolismo , Animales , Antivirales/clasificación , Antivirales/farmacología , Betacoronavirus/genética , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidad , COVID-19 , Chlorocebus aethiops , Clonación Molecular , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Evaluación Preclínica de Medicamentos , Células HEK293 , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Inmunidad Innata , Espectrometría de Masas , Pandemias , Neumonía Viral/inmunología , Neumonía Viral/virología , Unión Proteica , Biosíntesis de Proteínas/efectos de los fármacos , Dominios Proteicos , Mapeo de Interacción de Proteínas , Receptores sigma/metabolismo , SARS-CoV-2 , Proteínas Ligasas SKP Cullina F-box/metabolismo , Células Vero , Proteínas Virales/genética , Tratamiento Farmacológico de COVID-19RESUMEN
Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.
Asunto(s)
Aedes/virología , Infecciones por Alphavirus/etiología , Infecciones por Alphavirus/transmisión , Alphavirus/patogenicidad , Evolución Biológica , Estado Nutricional , Obesidad/virología , Infecciones por Alphavirus/patología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Mosquitos Vectores/virología , Obesidad/patología , Virulencia , Replicación ViralRESUMEN
The chikungunya virus (CHIKV) is transmitted by female Aedes aegypti and Aedes albopictus mosquitoes, mostly present in (sub)tropical regions. No antivirals are available to treat CHIKV infections. If antiviral drugs are proven efficient to treat CHIKV-infected patients, it will be pivotal to determine whether drug-resistant viruses can be transmitted from one human to another by their mosquito vectors. We orally infected Aedes aegypti mosquitoes with a blood meal containing wild-type or drug-resistant CHIKV variants (i.e., MADTPres CHIKV, with mutation in the nsP1 gene, and T-705res CHIKV, with mutation in the RNA-dependent RNA polymerase [RdRp] gene). Viral loads were quantified in bodies (infection), heads (dissemination), and saliva (transmission) of individual mosquitoes. The infection rate of the resistant viruses was similar to that of the wild-type virus. However, the dissemination of T-705res CHIKV was markedly decreased compared to wild-type and MADTPres CHIKV. Furthermore, T-705res CHIKV was only transmitted in the saliva at day 20 postinfection (p.i.), whereas transmission of wild-type CHIKV was observed at day 3 p.i. The attenuated phenotype of the T-705res virus was confirmed in mosquito cell culture, whereas the replication fitness in Vero cells was similar to that of the wild type. In bodies and heads of mosquitoes infected with the resistant variants, the resistant phenotype and genotype were retained. Also in the saliva, the resistant genotype of MADTPres CHIKV was maintained. Our results illustrate that the fitness of drug-resistant variants should be evaluated in both hosts to be able to select antiviral drugs with a limited risk for the spread of drug resistance by mosquitoes.IMPORTANCE Because of its global reemergence and unusual morbidities associated with infection, the chikungunya virus (CHIKV) has become a substantial public health problem. However, no antivirals are currently available to treat CHIKV infections. If antiviral drugs will prove to be efficient to treat CHIKV-infected patients, it will be essential to understand if drug-resistant viruses can be transmitted from one human to another by the mosquito. We therefore orally infected Aedes mosquitoes with drug-resistant CHIKV variants and determined the replication and transmission levels. One of the antiviral drug-resistant CHIKV variants could efficiently replicate and disseminate in both laboratory and field-collected mosquitoes. In addition, this variant retained its drug-resistant genotype in the saliva. In contrast, the other drug-resistant variant was markedly attenuated in mosquitoes. Our results illustrate that extra caution for drug resistance should be considered when developing an antiarbovirus antiviral in order to minimize the risk of spreading drug resistance by mosquitoes.