RESUMEN
Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.
Asunto(s)
Farmacorresistencia Fúngica , Antagonistas del Ácido Fólico , Metotrexato , Mutación , Pneumocystis carinii , Tetrahidrofolato Deshidrogenasa , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Pneumocystis carinii/genética , Pneumocystis carinii/enzimología , Pneumocystis carinii/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Farmacorresistencia Fúngica/genética , Metotrexato/farmacología , Regulación Alostérica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de los fármacos , Humanos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Dominio Catalítico/genéticaRESUMEN
Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein-protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions for >11 000 bait-prey pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.
Asunto(s)
Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae , Bioensayo , Humanos , Proteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Dengue is the most prevalent mosquito-borne viral disease. It is caused by the infection of any of the four dengue virus (DENV) serotypes DENV-1 to DENV-4. The DENV non-structural glycoprotein 1 (NS1) plays an important role in virus replication and the immunopathogenesis of virus infection. The NS1 protein has been identified as both a cell-associated homodimer and a soluble secreted lipoprotein nanoparticle. The nature of the residues at positions NS1-272 and NS1-324 in the ß-ladder domain may have an effect on the biological behaviors of DENV-2 NS1 protein in human hepatoma Huh7 cells. The stability of the NS1 protein from the Reunion 2018 DENV-2 strain was affected by the presence of lysine residues at positions 272 and 324. In the present study, we evaluated the impact of mutations into lysine at positions 272 and 324 on recombinant NS1 protein from the DES-14 DENV-2 strain bearing arginine residue on these two positions. The DES-14 NS1 protein mutant bearing a lysine at position 324 was deficient in protein stability and secretion compared to wild-type protein. The defect in the DES-14 NS1 protein mutant was associated to oxidative stress and pro-inflammatory cytokine activation in Huh7 cells. The ubiquitin-proteasome proteolytic pathway might play a key role in the stability of DENV-2 protein bearing a lysine residue at position 324.
RESUMEN
Mosquito-borne dengue disease is caused by the dengue virus serotype-1 to serotype-4. The contemporary dengue outbreaks in the southwestern Indian ocean coincided with the widespread of dengue virus serotype 2 genotype II (Cosmopolitan), including epidemic viral strains DES-14 and RUN-18 isolated in Dar es Salaam (Tanzania) in 2014 and La Reunion Island (France) in 2018, respectively. Heterodimeric interaction between prM (intracellular precursor of surface structural M protein) and envelope E proteins is required during the initial stage of dengue virus assembly. Amino acid 127 of DES-14 prM protein (equivalent to M36) has been identified as an infrequent valine whereas RUN-18 has a common isoleucine. In the present study, we examined the effect of M-I36V mutation on the expression of a recombinant RUN-18 E protein co-expressed with prM in human epithelial A549 cells. The M ectodomain of dengue virus serotype 2 embeds a pro-apoptotic peptide referred as D2AMP. The impact of M-I36V mutation on the death-promoting capability of D2AMP was assessed in A549 cells. We showed that valine at position M36 affects expression of recombinant RUN-18 E protein and potentiates apoptosis-inducing activity of D2AMP. We propose that the nature of M residue 36 influences the virological characteristics of dengue 2 M and E proteins belonging to genotype II that contributes to global dengue burden.
Asunto(s)
Virus del Dengue , Dengue , Animales , Humanos , Virus del Dengue/genética , Serogrupo , Tanzanía/epidemiología , GenotipoRESUMEN
Cell death by apoptosis is a major cellular response in the control of tissue homeostasis and as a defense mechanism in the case of cellular aggression such as an infection. Cell self-destruction is part of antiviral responses, aimed at limiting the spread of a virus. Although it may contribute to the deleterious effects in infectious pathology, apoptosis remains a key mechanism for viral clearance and the resolution of infection. The control mechanisms of cell death processes by viruses have been extensively studied. Apoptosis can be triggered by different viral determinants through different pathways as a result of virally induced cell stresses and innate immune responses. Zika virus (ZIKV) induces Zika disease in humans, which has caused severe neurological forms, birth defects, and microcephaly in newborns during the last epidemics. ZIKV also surprised by revealing an ability to persist in the genital tract and in semen, thus being sexually transmitted. Mechanisms of diverting antiviral responses such as the interferon response, the role of cytopathic effects and apoptosis in the etiology of the disease have been widely studied and debated. In this review, we examined the interplay between ZIKV infection of different cell types and apoptosis and how the virus deals with this cellular response. We illustrate a duality in the effects of ZIKV-controlled apoptosis, depending on whether it occurs too early or too late, respectively, in neuropathogenesis, or in long-term viral persistence. We further discuss a prospective role for apoptosis in ZIKV-related therapies, and the use of ZIKV as an oncolytic agent.
Asunto(s)
Apoptosis/fisiología , Infección por el Virus Zika/metabolismo , Virus Zika/fisiología , Animales , Antivirales/uso terapéutico , Muerte Celular/fisiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/inmunología , Interferones/uso terapéutico , Microcefalia/virología , Fenómenos Fisiológicos de los Virus/inmunología , Replicación Viral/fisiología , Virus Zika/genética , Virus Zika/patogenicidad , Infección por el Virus Zika/virologíaRESUMEN
Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Virus del Dengue/química , Virus del Dengue/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Reacciones Cruzadas/inmunología , Cristalografía por Rayos X , Virus del Dengue/clasificación , Epítopos/química , Epítopos/inmunología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Conformación Proteica , Multimerización de Proteína , Solubilidad , Especificidad de la Especie , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
BACKGROUND: Polypharmacy is common among older adults and it represents a public health concern, due to the negative health impacts potentially associated with the use of several medications. However, the large number of medication combinations and sequences of use makes it complicated for traditional statistical methods to predict which therapy is genuinely associated with health outcomes. The project aims to use artificial intelligence (AI) to determine the quality of polypharmacy among older adults with chronic diseases in the province of Québec, Canada. METHODS: We will use data from the Quebec Integrated Chronic Disease Surveillance System (QICDSS). QICDSS contains information about prescribed medications in older adults in Quebec collected over 20 years. It also includes diagnostic codes and procedures, and sociodemographic data linked through a unique identification number for each individual. Our research will be structured around three interconnected research axes: AI, Health, and Law&Ethics. The AI research axis will develop algorithms for finding frequent patterns of medication use that correlate with health events, considering data locality and temporality (explainable AI or XAI). The Health research axis will translate these patterns into polypharmacy indicators relevant to public health surveillance and clinicians. The Law&Ethics axis will assess the social acceptability of the algorithms developed using AI tools and the indicators developed by the Heath axis and will ensure that the developed indicators neither discriminate against any population group nor increase the disparities already present in the use of medications. DISCUSSION: The multi-disciplinary research team consists of specialists in AI, health data, statistics, pharmacy, public health, law, and ethics, which will allow investigation of polypharmacy from different points of view and will contribute to a deeper understanding of the clinical, social, and ethical issues surrounding polypharmacy and its surveillance, as well as the use of AI for health record data. The project results will be disseminated to the scientific community, healthcare professionals, and public health decision-makers in peer-reviewed publications, scientific meetings, and reports. The diffusion of the results will ensure the confidentiality of individual data.
Asunto(s)
Inteligencia Artificial , Polifarmacia , Anciano , Enfermedad Crónica , Análisis de Datos , Humanos , QuebecRESUMEN
Zika virus (ZIKV) is an emerging mosquito-borne flavivirus considered as a threat to human health due to large epidemics and serious clinical outcomes such as microcephaly in new-borns. Like all flaviviruses, ZIKV relies on the cellular machinery to complete its viral cycle, with the endoplasmic reticulum (ER) being the critical site of viral replication factories. The sudden high protein load in the ER induces an ER stress to which the cell responds with an appropriate unfolded protein response (UPR) in an attempt to restore its disturbed homeostasis. When the restoration fails, the cell signalling leads to a programmed cell death by apoptosis with the upregulation of the UPR-induced C/EBP homologous protein (CHOP) which acts as the main trigger for this fatal outcome. Our previous studies have shown the ability of ZIKV to manipulate various cellular responses in order to optimize virus production. ZIKV is able to delay apoptosis to its benefit and although ER stress is induced, the UPR is not complete. Here we discovered that ZIKV impairs the expression of CHOP/DDIT3, the main factor responsible of ER-stress driven apoptosis. Surprisingly, the mechanism does not take place at the transcriptional level but at the translational level.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Factor de Transcripción CHOP/metabolismo , Transcripción Genética , Infección por el Virus Zika/metabolismo , Virus Zika/metabolismo , Células A549 , HumanosRESUMEN
Mosquito-borne Zika virus (ZIKV) became a real threat to human health due to the lack of vaccine and effective antiviral treatment. The virus has recently been responsible for a global outbreak leading to millions of infected cases. ZIKV complications were highlighted in adults with Guillain-Barré syndrome and in newborns with increasing numbers of congenital disorders ranging from mild developmental delays to fatal conditions. The ability of ZIKV to establish a long-term infection in diverse organs including the kidneys has been recently documented but the consequences of such a viral infection are still debated. Our study aimed to determine whether the efficiency of ZIKV growth in kidney cells relates to glucose concentration. Human kidney HK-2 cells were infected with different ZIKV strains in presence of normal and high glucose concentrations. Virological assays showed a decrease in viral replication without modifying entry steps (viral binding, internalization, fusion) under high glucose conditions. This decrease replication was associated with a lower virus progeny and increased cell viability when compared to ZIKV-infected HK-2 cells in normal glucose concentration. In conclusion, we showed for the first time that an elevated glucose level influences ZIKV replication level with an effect on kidney cell survival.
Asunto(s)
Glucosa/farmacología , Riñón/efectos de los fármacos , Replicación Viral , Infección por el Virus Zika/prevención & control , Virus Zika/crecimiento & desarrollo , Células Cultivadas , Humanos , Riñón/virología , Edulcorantes/farmacología , Acoplamiento Viral , Virus Zika/efectos de los fármacos , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/virologíaRESUMEN
La Reunion island in the South West Indian Ocean is now endemic for dengue following the introduction of dengue virus serotype 2 (DENV-2) cosmopolitan-I genotype in 2017. DENV-2 infection causes a wide spectrum of clinical manifestations ranging from flu-like disease to severe dengue. The nonstructural glycoprotein 1 (NS1) has been identified as playing a key role in dengue disease severity. The intracellular NS1 exists as a homodimer, whereas a fraction is driven towards the plasma membrane or released as a soluble hexameric protein. Here, we characterized the NS1 glycoproteins from clinical isolates DES-14 and RUN-18 that were collected during the DENV-2 epidemics in Tanzania in 2014 and La Reunion island in 2018, respectively. In relation to hepatotropism of the DENV, expression of recombinant DES-14 NS1 and RUN-18 NS1 glycoproteins was compared in human hepatoma Huh7 cells. We observed that RUN-18 NS1 was poorly stable in Huh7 cells compared to DES-14 NS1. The instability of RUN-18 NS1 leading to a low level of NS1 secretion mostly relates to lysine residues on positions 272 and 324. Our data raise the issue of the consequences of a defect in NS1 stability in human hepatocytes in relation to the major role of NS1 in the pathogenesis of the DENV-2 infection.
Asunto(s)
Virus del Dengue/metabolismo , Dengue/epidemiología , Dengue/metabolismo , Epidemias , Genotipo , Lisina/química , Proteínas no Estructurales Virales/química , Sustitución de Aminoácidos , Antígenos Virales/química , Antígenos Virales/genética , Línea Celular Tumoral , Dengue/virología , Células HEK293 , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/química , Reunión/epidemiología , Serogrupo , Tanzanía/epidemiología , Transfección , Proteínas no Estructurales Virales/genéticaRESUMEN
The recent emergence of Zika virus (ZIKV) in Brazil and the increasing resistance developed by pathogenic bacteria to nearly all existing antibiotics should be taken as a wakeup call for the international authority as this represents a risk for global public health. The lack of antiviral drugs and effective antibiotics on the market triggers the need to search for safe therapeutics from medicinal plants to fight viral and microbial infections. In the present study, we investigated whether a mangrove plant, Bruguiera gymnorhiza (L.) Lam. (B. gymnorhiza) collected in Mauritius, possesses antimicrobial and antibiotic potentiating abilities and exerts anti-ZIKV activity at non-cytotoxic doses. Microorganisms Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70603, methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Salmonella enteritidis ATCC 13076, Sarcina lutea ATCC 9341, Proteus mirabilis ATCC 25933, Bacillus cereus ATCC 11778 and Candida albicans ATCC 26555 were used to evaluate the antimicrobial properties. Ciprofloxacin, chloramphenicol and streptomycin antibiotics were used for assessing antibiotic potentiating activity. ZIKVMC-MR766NIID (ZIKVGFP) was used for assessing anti-ZIKV activity. In silico docking (Autodock 4) and ADME (SwissADME) analyses were performed on collected data. Antimicrobial results revealed that Bruguiera twig ethyl acetate (BTE) was the most potent extract inhibiting the growth of all nine microbes tested, with minimum inhibitory concentrations ranging from 0.19-0.39 mg/mL. BTE showed partial synergy effects against MRSA and Pseudomonas aeruginosa when applied in combination with streptomycin and ciprofloxacin, respectively. By using a recombinant ZIKV-expressing reporter GFP protein, we identified both Bruguiera root aqueous and Bruguiera fruit aqueous extracts as potent inhibitors of ZIKV infection in human epithelial A549 cells. The mechanisms by which such extracts prevented ZIKV infection are linked to the inability of the virus to bind to the host cell surface. In silico docking showed that ZIKV E protein, which is involved in cell receptor binding, could be a target for cryptochlorogenic acid, a chemical compound identified in B. gymnorhiza. From ADME results, cryptochlorogenic acid is predicted to be not orally bioavailable because it is too polar. Scientific data collected in this present work can open a new avenue for the development of potential inhibitors from B. gymnorhiza to fight ZIKV and microbial infections in the future.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Antivirales/farmacología , Extractos Vegetales/farmacología , Rhizophoraceae/química , Virus Zika/crecimiento & desarrollo , Antibacterianos/química , Antifúngicos/química , Antivirales/química , Brasil , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Simulación por Computador , Sinergismo Farmacológico , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/crecimiento & desarrollo , Mauricio , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Extractos Vegetales/química , Proteus mirabilis/efectos de los fármacos , Proteus mirabilis/crecimiento & desarrollo , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Virus Zika/efectos de los fármacosRESUMEN
BACKGROUND: Radiotherapy and brachytherapy are common treatments for localized prostate cancer (PCa). However, very few studies evaluated the association of variations in DNA damage response genes and treatment outcomes and toxicity in brachytherapy-treated patients. PURPOSE: To evaluate the association of inherited germline variations in DNA repair-associated genes with tumor control and treatment toxicity in patients treated with low-dose-rate prostate brachytherapy (LDRB). MATERIAL AND METHODS: The cohort consists of 475 I-125 LDRB patients with a median follow-up of 51 months after seed implantation. Patients were genotyped for 215 haplotype tagging single nucleotide variations (htSNPs) in 29 candidate genes of DNA damage response and repair pathways. Their association with biochemical recurrence (BCR) was assessed using Cox regression models and Kaplan-Meier survival curves. Linear regressions and analysis of covariance (ANCOVA) between early and late International Prostate Symptom Score (IPSS) with htSNPs were used to evaluate the association with urinary toxicity. RESULTS: After adjustment for the established risk factors, six htSNPs in five genes were found to be significantly associated with an altered risk of BCR, with adjusted hazard ratios (HRadj. ) ranging between 3.6 and 11.1 (P < .05). Compared to carriers of the ERCC3 rs4150499C allele, patients homozygous for the T allele (n = 22) had a significant higher risk of BCR with a HR of 11.13 (IC95 = 3.9-32.0; P < .0001; q < 0.001). The Kaplan-Meier survival curve revealed a mean BCR-free survival time reduced from 213 ± 7 to 99 ± 12 months (log-rank P < .0001) for homozygous T carriers compare to noncarriers. For late IPSS (>6 months after treatment), htSNP rs6544990 from MSH2 showed a statistically significant b-coefficient of 1.85 ± 0.52 (P < .001; q < 0.1). Homozygous carriers of the MSH2 rs6544990C allele (n = 62) had a mean late IPSS 3.6 points higher than patients homozygous for the A allele (n = 132). This difference was significant when tested by ANCOVA using pretreatment IPSS as a covariate (P < .01). CONCLUSIONS: This study suggests an association of the intronic variants of the DNA nucleotide excision repair ERCC3 and DNA mismatch repair MSH2 genes with elevated risk of BCR and late urinary toxicity respectively after LDRB. Further validation is required before translational clinical advances.
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Braquiterapia/efectos adversos , Braquiterapia/métodos , Reparación del ADN/genética , Radioisótopos de Yodo/administración & dosificación , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/radioterapia , Mutación de Línea Germinal , Humanos , Radioisótopos de Yodo/efectos adversos , Masculino , Enfermedades Urogenitales Masculinas/etiología , Enfermedades Urogenitales Masculinas/genética , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Traumatismos por Radiación/etiología , Traumatismos por Radiación/genéticaRESUMEN
Iterative reconstruction has been proven to be an effective tool for low-dose computed tomography imaging. However, this technology is currently not available in commercial diagnostic maxillofacial cone beam CT. For this technical note, an iterative reconstruction technique was applied to cone beam CT raw data of two maxillofacial clinical cases to explore its potential for dose reduction and metal artifact reduction. Low-dose imaging was emulated by using only fractions of the clinical projection dataset. The reconstruction algorithms tested were filtered backprojection (FBP) as a reference method, and a total variation minimization (TV) regularized ordered subsets convex (OSC-TV) method as the iterative technique. Upon qualitative examination, the OSC-TV technique was found to conserve most diagnostic information using half the projections. Test images have also shown that at 1/4 of the projections, OSC-TV was more robust than FBP with respect to streaking and metal artifacts.
Asunto(s)
Tomografía Computarizada de Haz Cónico/métodos , Seno Maxilar/diagnóstico por imagen , Interpretación de Imagen Radiográfica Asistida por Computador/métodos , Trastornos de la Articulación Temporomandibular/diagnóstico por imagen , Algoritmos , Artefactos , Niño , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay. We investigate the architecture of the RNA polymerases, the proteasome and the conserved oligomeric Golgi (COG) complexes in living cells and performed large-scale screens with these extended linkers. We show that longer linkers significantly improve the detection of protein-protein interactions and allow to measure interactions further in space than the standard ones. We identify new interactions, for instance between the retromer complex and proteins related to autophagy and endocytosis. Longer linkers thus contribute an enhanced additional tool to the existing toolsets for the detection and measurements of protein-protein interactions and protein proximity in living cells.
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Mapeo de Interacción de Proteínas/métodos , Tetrahidrofolato Deshidrogenasa/metabolismo , Bioensayo , Escherichia coli/genética , Tetrahidrofolato Deshidrogenasa/genética , Levaduras/genéticaRESUMEN
Mosquito-borne Zika virus (ZIKV) is an emerging flavivirus of medical concern associated with neurological disorders. ZIKV utilizes apoptosis as a mechanism of cell killing. The structural M protein may play a role in flavivirus-induced apoptosis. The death-promoting capability of M has been restricted to an oligopeptide representing the residues M-32/40. Here, we evaluated the apoptosis inducing ability of the residues M-31/41 of ZIKV. The ZIKV M oligopeptide was associated to a soluble form of GFP (sGFP) and the resulting sGFP-M31/41 construct was assessed in Huh7 cells. Expression of sGFP-M31/41 can trigger apoptosis in Huh7 cells through caspase-3/7 activation. The translocation of sGFP-M31/41 in the endoplasmic reticulum was a prerequisite for apoptosis induction. The residues M-33/35/38 may play a critical role in the death-promoting activity of sGFP-M31/41. The effect of ZIKV M oligopeptide defined as ZAMP (for Zika Apoptosis M Peptide) on expression of a tumor-associated antigen was assayed on megakaryocyte-potentiating factor (MPF). Expression of MPF-ZAMP construct resulted in caspase-associated apoptosis activation in A549 and Huh7 cells. ZIKV has been proposed as an oncolytic virus for cancer therapy. The ability of the Zika M oligopeptide to confer death-promoting capability to MPF opens up attractive perspectives for ZAMP as an innovative anticancer agent.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Apoptosis , Proteínas Ligadas a GPI/metabolismo , Oligopéptidos/metabolismo , Proteínas de la Matriz Viral/química , Virus Zika/química , Células A549 , Antígenos de Neoplasias/genética , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Ligadas a GPI/genética , Células HEK293 , Humanos , Mesotelina , Oligopéptidos/química , Oligopéptidos/genéticaRESUMEN
The author wishes to make the following correction to this paper [...].
RESUMEN
The mosquito-borne viruses dengue (DENV) and Zika (ZIKV) viruses are two medically important pathogens in tropical and subtropical regions of the world. There is an urgent need of therapeutics against DENV and ZIKV, and medicinal plants are considered as a promising source of antiviral bioactive metabolites. In the present study, we evaluated the ability of Phyllanthus phillyreifolius, an endemic medicinal plant from Reunion Island, to prevent DENV and ZIKV infection in human cells. At non-cytotoxic concentration in vitro, incubation of infected A549 cells with a P. phillyreifolius extract or its major active phytochemical geraniin resulted in a dramatic reduction of virus progeny production for ZIKV as well as four serotypes of DENV. Virological assays showed that P. phillyreifolius extract-mediated virus inhibition relates to a blockade in internalization of virus particles into the host cell. Infectivity studies on ZIKV showed that both P. phillyreifolius and geraniin cause a loss of infectivity of the viral particles. Using a zebrafish model, we demonstrated that administration of P. phillyreifolius and geraniin has no effect on zebrafish locomotor activity while no morbidity nor mortality was observed up to 5 days post-inoculation. Thus, P. phillyreifolius could act as an important source of plant metabolite geraniin which is a promising antiviral compound in the fight against DENV and ZIKV.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Glucósidos/farmacología , Taninos Hidrolizables/farmacología , Phyllanthus/química , Fitoquímicos/farmacología , Internalización del Virus/efectos de los fármacos , Virus Zika/efectos de los fármacos , Células A549 , Animales , Antivirales/aislamiento & purificación , Línea Celular Tumoral , Chlorocebus aethiops , Virus del Dengue/crecimiento & desarrollo , Glucósidos/aislamiento & purificación , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Taninos Hidrolizables/aislamiento & purificación , Fitoquímicos/aislamiento & purificación , Extractos Vegetales/química , Plantas Medicinales , Reunión , Células Vero , Pez Cebra , Virus Zika/crecimiento & desarrolloRESUMEN
Interferon-induced viperin (VP) was identified as playing an important role in the innate immune response against Zika virus (ZIKV). The 361 amino acid long human VP protein comprises of a highly conserved C-terminal region, which has been associated with VP antiviral properties against ZIKV. In the present study, we sought to determine whether the very last C-terminal amino-acid residues of VP might play a role in VP-mediated ZIKV inhibition. To address this issue, a recombinant human viperin (rVPwt) was overexpressed by transfection in human epithelial A549 cells. We confirmed that transient overexpression of rVPwt prior to ZIKV infection dramatically reduced viral replication in A549 cells. Deletion of the last 17 C-terminal amino acids of VP resulted in a higher expression level of mutant protein compared to wild-type VP. Mutational analysis revealed that residue substitution at positions 356 to 360 with five alanine led to the same phenotype. The charged residues Asp356, Lys358, and Asp360 were then identified to play a role in the weak level of VPwt protein in A549 cells. Mutant VP bearing the D360A substitution partially rescued ZIKV growth in A549 cells. Remarkably, a single Lys-to-Arg substitution at position 358 was sufficient to abrogate VP antiviral activity against ZIKV. In conclusion, our study showed that Asp356, Lys358, and Asp360 may have an influence on biochemical properties of VP. Our major finding was that Lys358 was a key amino-acid in VP antiviral properties against ZIKV.
Asunto(s)
Sustitución de Aminoácidos , Antivirales/farmacología , Proteínas Mutantes/metabolismo , Proteínas/genética , Virus Zika/efectos de los fármacos , Células A549 , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Humanos , Proteínas Mutantes/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes/farmacología , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Zika virus (ZIKV) and Dengue virus (DENV) are mosquito-borne viruses of the Flavivirus genus that could cause congenital microcephaly and hemorrhage, respectively, in humans, and thus present a risk to global public health. A preventive vaccine against ZIKV remains unavailable, and no specific antiviral drugs against ZIKV and DENV are licensed. Medicinal plants may be a source of natural antiviral drugs which mostly target viral entry. In this study, we evaluate the antiviral activity of Doratoxylum apetalum, an indigenous medicinal plant from the Mascarene Islands, against ZIKV and DENV infection. Our data indicated that D. apetalum exhibited potent antiviral activity against a contemporary epidemic strain of ZIKV and clinical isolates of four DENV serotypes at non-cytotoxic concentrations in human cells. Time-of-drug-addition assays revealed that D. apetalum extract acts on ZIKV entry by preventing the internalisation of virus particles into the host cells. Our data suggest that D. apetalum-mediated ZIKV inhibition relates to virus particle inactivation. We suggest that D. apetalum could be a promising natural source for the development of potential antivirals against medically important flaviviruses.
Asunto(s)
Antivirales/farmacología , Virus del Dengue/efectos de los fármacos , Extractos Vegetales/farmacología , Sapindaceae/química , Virus Zika/efectos de los fármacos , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Plantas Medicinales/química , Células VeroRESUMEN
The recent emergence and re-emergence of viral infections transmitted by vectors, such as the Zika virus (ZIKV) and Dengue virus (DENV), is a cause for international concern. These highly pathogenic arboviruses represent a serious health burden in tropical and subtropical areas of the world. Despite the high morbidity and mortality associated with these viral infections, antiviral therapies are missing. Medicinal plants have been widely used to treat various infectious diseases since millenaries. Several compounds extracted from plants exhibit potent effects against viruses in vitro, calling for further investigations regarding their efficacy as antiviral drugs. Here, we demonstrate that an extract from Psiloxylon mauritianum, an endemic medicinal plant from Reunion Island, inhibits the infection of ZIKV in vitro without exhibiting cytotoxic effects. The extract was active against different ZIKV African and Asian strains, including an epidemic one. Time-of-drug-addition assays revealed that the P. mauritianum extract interfered with the attachment of the viral particles to the host cells. Importantly, the P. mauritianum extract was also able to prevent the infection of human cells by four dengue virus serotypes. Due to its potency and ability to target ZIKV and DENV particles, P. mauritianum may be of value for identifying and characterizing antiviral compounds to fight medically-important flaviviruses.