RESUMEN
The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlighted a critical need to discover more effective antivirals. While therapeutics for SARS-CoV-2 exist, its nonstructural protein 13 (Nsp13) remains a clinically untapped target. Nsp13 is a helicase responsible for unwinding double-stranded RNA during viral replication and is essential for propagation. Like other helicases, Nsp13 has two active sites: a nucleotide binding site that hydrolyzes nucleoside triphosphates (NTPs) and a nucleic acid binding channel that unwinds double-stranded RNA or DNA. Targeting viral helicases with small molecules, as well as the identification of ligand binding pockets, have been ongoing challenges, partly due to the flexible nature of these proteins. Here, we use a virtual screen to identify ligands of Nsp13 from a collection of clinically used drugs. We find that a known ion channel inhibitor, IOWH-032, inhibits the dual ATPase and helicase activities of SARS-CoV-2 Nsp13 at low micromolar concentrations. Kinetic and binding assays, along with computational and mutational analyses, indicate that IOWH-032 interacts with the RNA binding interface, leading to displacement of nucleic acid substrate, but not bound ATP. Evaluation of IOWH-032 with microbial helicases from other superfamilies reveals that it is selective for coronavirus Nsp13. Furthermore, it remains active against mutants representative of observed SARS-CoV-2 variants. Overall, this work provides a new inhibitor for Nsp13 and provides a rationale for a recent observation that IOWH-032 lowers SARS-CoV-2 viral loads in human cells, setting the stage for the discovery of other potent viral helicase modulators.
Asunto(s)
Antivirales , Reposicionamiento de Medicamentos , SARS-CoV-2 , Proteínas no Estructurales Virales , SARS-CoV-2/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/farmacología , Antivirales/química , Humanos , ARN Helicasas/metabolismo , ARN Helicasas/antagonistas & inhibidores , COVID-19/virología , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/química , Betacoronavirus/efectos de los fármacos , Tratamiento Farmacológico de COVID-19 , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , MetiltransferasasRESUMEN
Around the world, rollout of COVID-19 vaccines has been used as a strategy to end COVID-19-related restrictions and the pandemic. Several COVID-19 vaccine platforms have successfully protected against severe SARS-CoV-2 infection and subsequent deaths. Here, we compared humoral and cellular immunity in response to either infection or vaccination. We examined SARS-CoV-2 spike-specific immune responses from Pfizer/BioNTech BNT162b2, Moderna mRNA-1273, Janssen Ad26.COV2.S, and SARS-CoV-2 infection approximately 4 months post-exposure or vaccination. We found that these three vaccines all generate relatively similar immune responses and elicit a stronger response than natural infection. However, antibody responses to recent viral variants are diminished across all groups. The similarity of immune responses from the three vaccines studied here is an important finding in maximizing global protection as vaccination campaigns continue.
RESUMEN
As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.
Asunto(s)
Infecciones por VIH , VIH-1 , Teorema de Bayes , Infecciones por VIH/metabolismo , VIH-1/genética , Humanos , Proteoma/metabolismo , ProteómicaRESUMEN
SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA ≈ EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5' position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.
Asunto(s)
Amilorida/farmacología , Tratamiento Farmacológico de COVID-19 , Proteínas de la Envoltura de Coronavirus/metabolismo , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Amilorida/farmacocinética , Animales , Antivirales/farmacología , Sitios de Unión/efectos de los fármacos , COVID-19/virología , Chlorocebus aethiops , Proteínas de la Envoltura de Coronavirus/química , Humanos , Canales Iónicos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Dominios Proteicos , Células Vero , Ensamble de Virus/efectos de los fármacosRESUMEN
A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.
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Antígenos CD/genética , Interacciones Huésped-Patógeno/genética , Factores Reguladores del Interferón/genética , Interferón Tipo I/genética , SARS-CoV-2/genética , Proteínas Virales/genética , Animales , Antígenos CD/química , Antígenos CD/inmunología , Sitios de Unión , Línea Celular Tumoral , Chlorocebus aethiops , Retículo Endoplásmico/genética , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/virología , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Regulación de la Expresión Génica , Aparato de Golgi/genética , Aparato de Golgi/inmunología , Aparato de Golgi/virología , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Factores Reguladores del Interferón/clasificación , Factores Reguladores del Interferón/inmunología , Interferón Tipo I/inmunología , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/inmunología , Transducción de Señal , Células Vero , Proteínas Virales/química , Proteínas Virales/inmunología , Internalización del Virus , Liberación del Virus/genética , Liberación del Virus/inmunología , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
A deficient interferon response to SARS-CoV-2 infection has been implicated as a determinant of severe COVID-19. To identify the molecular effectors that govern interferon control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human interferon stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors that inhibited viral entry, nucleic acid binding proteins that suppressed viral RNA synthesis, and a highly enriched cluster of ER and Golgi-resident ISGs that inhibited viral translation and egress. These included the type II integral membrane protein BST2/tetherin, which was found to impede viral release, and is targeted for immune evasion by SARS-CoV-2 Orf7a protein. Overall, these data define the molecular basis of early innate immune control of viral infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.
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In our daily lives, we consume foods that have been transported, stored, prepared, cooked, or otherwise processed by ourselves or others. Food storage and preparation have drastic effects on the chemical composition of foods. Untargeted mass spectrometry analysis of food samples has the potential to increase our chemical understanding of these processes by detecting a broad spectrum of chemicals. We performed a time-based analysis of the chemical changes in foods during common preparations, such as fermentation, brewing, and ripening, using untargeted mass spectrometry and molecular networking. The data analysis workflow presented implements an approach to study changes in food chemistry that can reveal global alterations in chemical profiles, identify changes in abundance, as well as identify specific chemicals and their transformation products. The data generated in this study are publicly available, enabling the replication and re-analysis of these data in isolation, and serve as a baseline dataset for future investigations.
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Bebidas/análisis , Análisis de los Alimentos , Manipulación de Alimentos , Espectrometría de Masas , Metabolómica , Fermentación , Flujo de TrabajoRESUMEN
Since the re-emergence of Zika virus in 2014 and subsequent association with microcephaly, much work has focused on the development of a vaccine to halt its spread throughout the world. The mosquito vector that transmits this virus is widespread and responsible for the spread of other arboviridae including Dengue. Current diagnostic methods rely on serologic testing that are complicated by cross reactivity and therefore unable to distinguish Zika from Dengue infection in the absence of virus isolation. We performed an in silico analysis to identify potential epitopes that may stimulate a unique T-lymphocyte response to distinguish prior infection with Zika or Dengue. From this analysis, we not only identified epitopes unique to Zika and Dengue, but also identified epitopes unique to each Dengue serotype. These peptides contribute to a pool of peptides identified for vaccine development that can be tested in vitro to confirm immunogenicity, absence of homology and global population coverage. The current lack of accurate diagnostic testing hampers our ability to understand the scope of the epidemic, implications for vaccine implementation and complications related to monoinfection and co-infection with these two closely related viruses.
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Dengue/diagnóstico , Epítopos/inmunología , Fragmentos de Péptidos/inmunología , Pruebas Serológicas/métodos , Proteínas no Estructurales Virales/inmunología , Infección por el Virus Zika/diagnóstico , Dengue/inmunología , Dengue/virología , Virus del Dengue/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Virus Zika/inmunología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.
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Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Centro Germinal/inmunología , Anticuerpos Anti-VIH/uso terapéutico , Infecciones por VIH/terapia , VIH-1/inmunología , Animales , Células Cultivadas , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Centro Germinal/virología , Infecciones por VIH/inmunología , Humanos , Inmunización , Inyecciones Subcutáneas , Primates , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
First discovered in 1993, microRNAs (miRNAs) have been one of the hottest research areas over the past two decades. Oftentimes, miRNAs levels are found to be dysregulated in cancer patients. The potential use of miRNAs in cancer therapies is an emerging and promising field, with research finding miRNAs to play a role in cancer initiation, tumor growth, and metastasis. Therefore, miRNAs could become an integral part from cancer diagnosis to treatment in future. This review aims to examine current novel research work on the potential roles of miRNAs in cancer therapies, while also discussing several current challenges and needed future research.
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Biomarcadores de Tumor/biosíntesis , Regulación Neoplásica de la Expresión Génica , MicroARNs/biosíntesis , Neoplasias/genética , Biomarcadores de Tumor/genética , Resistencia a Antineoplásicos/genética , Humanos , MicroARNs/genética , Terapia Molecular Dirigida , Neoplasias/diagnóstico , Neoplasias/terapiaRESUMEN
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and lacks effective targeted therapies. The microRNA-200 (miR-200) family is found to inhibit or promote breast cancer metastasis; however, the underlying mechanism is not well understood. This study was performed to investigate the effect and mechanism of miR-200b on TNBC metastasis and identify targets for developing more efficient treatment for TNBC. We found that miR-200 family expression levels are significantly lower in highly migratory TNBC cells and metastatic TNBC tumors than other types of breast cancer cells and tumors. Ectopically expressing a single member (miR-200b) of the miR-200 family drastically reduces TNBC cell migration and inhibits tumor metastasis in an orthotopic mouse mammary xenograft tumor model. We identified protein kinase Cα (PKCα) as a new direct target of miR-200b and found that PKCα protein levels are inversely correlated with miR-200b levels in 12 kinds of breast cancer cells. Inhibiting PKCα activity or knocking down PKCα levels significantly reduces TNBC cell migration. In contrast, forced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration and tumor metastasis. Further mechanistic studies revealed that PKCα downregulation by miR-200b results in a significant decrease of Rac1 activation in TNBC cells. These results show that loss of miR-200b expression plays a crucial role in TNBC aggressiveness and that miR-200b suppresses TNBC cell migration and tumor metastasis by targeting PKCα. Our findings suggest that miR-200b and PKCα may serve as promising therapeutic targets for metastatic TNBC.