RESUMEN
In this study, a previously undescribed cassane diterpenoid, named caesalpinin JF (1), along with two known cassane diterpenoids caesanine C (2) and tomocinol B (3), was isolated from 95% EtOH extract of the seeds of Caesalpinia sappan Linn. Additionally, three known compounds including pulcherrin R (4), syringaresinol-4'-O-ß-D-glucopyranoside (5) and kaempferol (6) were also identified. The structures of the isolated compounds were elucidated by comprehensive 1D and 2D NMR spectroscopic analyses. Additionally, electronic circular dichroism (ECD) calculation was used to identify the absolute structure of compound 1. Among the isolated compounds, compound 1 displayed a potent anti-neuroinflammation with an IC50 value of 9.87 ± 1.71 µM.
RESUMEN
Resistance genes (R genes) are a class of genes that are immune to a wide range of diseases and pests. In planta, NLR genes are essential components of the innate immune system. Currently, genes belonging to NLR family have been found in a number of plant species, but little is known in peach. Here, 286 NLR genes were identified on peach genome by using their homologous genes in Arabidopsis thaliana as queries. These 286 NLR genes contained at least one NBS domain and LRR domain. Phylogenetic and N-terminal domain analysis showed that these NLRs could be separated into four subfamilies (I-IV) and their promoters contained many cis-elements in response to defense and phytohormones. In addition, transcriptome analysis showed that 22 NLR genes were up-regulated after infected by Green Peach Aphid (GPA), and showed different expression patterns. This study clarified the NLR gene family and their potential functions in aphid resistance process. The candidate NLR genes might be useful in illustrating the mechanism of aphid resistance in peach.
Asunto(s)
Áfidos , Proteínas de Arabidopsis , Arabidopsis , Animales , Proteínas de Arabidopsis/genética , Áfidos/fisiología , Leucina/genética , Filogenia , Arabidopsis/genética , Nucleótidos/metabolismoRESUMEN
While emerging evidence highlights the significance of gut microbiome in gastrointestinal infectious diseases, treatments like Fecal Microbiota Transplantation (FMT) and probiotics are gaining popularity, especially for diarrhea patients. However, the specific role of the gut microbiome in different gastrointestinal infectious diseases remains uncertain. There is no consensus on whether gut modulation therapy is universally effective for all such infections. In this comprehensive review, we examine recent developments of the gut microbiome's involvement in several gastrointestinal infectious diseases, including infection of Helicobacter pylori, Clostridium difficile, Vibrio cholerae, enteric viruses, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa Staphylococcus aureus, Candida albicans, and Giardia duodenalis. We have also incorporated information about fungi and engineered bacteria in gastrointestinal infectious diseases, aiming for a more comprehensive overview of the role of the gut microbiome. This review will provide insights into the pathogenic mechanisms of the gut microbiome while exploring the microbiome's potential in the prevention, diagnosis, prediction, and treatment of gastrointestinal infections.
Asunto(s)
Enfermedades Gastrointestinales , Microbioma Gastrointestinal , Probióticos , Humanos , Trasplante de Microbiota Fecal , Probióticos/uso terapéutico , Enfermedades Gastrointestinales/terapia , Diarrea , Salmonella typhimuriumRESUMEN
Ebola (EBOV) and Marburg (MARV) are members of the Filoviridae family, which continue to emerge and cause sporadic outbreaks of hemorrhagic fever with high mortality rates. Filoviruses utilize their VP40 matrix protein to drive virion assembly and budding, in part, by recruitment of specific WW-domain-bearing host proteins via its conserved PPxY Late (L) domain motif. Here, we screened an array of 115 mammalian, bacterially expressed and purified WW-domains using a PPxY-containing peptide from MARV VP40 (mVP40) to identify novel host interactors. Using this unbiased approach, we identified Yes Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ) as novel mVP40 PPxY interactors. YAP and TAZ function as downstream transcriptional effectors of the Hippo signaling pathway that regulates cell proliferation, migration and apoptosis. We demonstrate that ectopic expression of YAP or TAZ along with mVP40 leads to significant inhibition of budding of mVP40 VLPs in a WW-domain/PPxY dependent manner. Moreover, YAP colocalized with mVP40 in the cytoplasm, and inhibition of mVP40 VLP budding was more pronounced when YAP was localized predominantly in the cytoplasm rather than in the nucleus. A key regulator of YAP nuclear/cytoplasmic localization and function is angiomotin (Amot); a multi-PPxY containing protein that strongly interacts with YAP WW-domains. Interestingly, we found that expression of PPxY-containing Amot rescued mVP40 VLP egress from either YAP- or TAZ-mediated inhibition in a PPxY-dependent manner. Importantly, using a stable Amot-knockdown cell line, we found that expression of Amot was critical for efficient egress of mVP40 VLPs as well as egress and spread of authentic MARV in infected cell cultures. In sum, we identified novel negative (YAP/TAZ) and positive (Amot) regulators of MARV VP40-mediated egress, that likely function in part, via competition between host and viral PPxY motifs binding to modular host WW-domains. These findings not only impact our mechanistic understanding of virus budding and spread, but also may impact the development of new antiviral strategies.
Asunto(s)
Filoviridae/fisiología , Marburgvirus/fisiología , Imitación Molecular , Proteínas Proto-Oncogénicas c-yes/metabolismo , Proteínas de la Matriz Viral/fisiología , Liberación del Virus , Angiomotinas , Sitios de Unión , Membrana Celular/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Dominios PDZ , Dominios Proteicos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The Ebola virus (EBOV) VP40 matrix protein (eVP40) orchestrates assembly and budding of virions in part by hijacking select WW-domain-bearing host proteins via its PPxY late (L)-domain motif. Angiomotin (Amot) is a multifunctional PPxY-containing adaptor protein that regulates angiogenesis, actin dynamics, and cell migration/motility. Amot also regulates the Hippo signaling pathway via interactions with the WW-domain-containing Hippo effector protein Yes-associated protein (YAP). In this report, we demonstrate that endogenous Amot is crucial for positively regulating egress of eVP40 virus-like particles (VLPs) and for egress and spread of authentic EBOV. Mechanistically, we show that ectopic YAP expression inhibits eVP40 VLP egress and that Amot co-expression rescues budding of eVP40 VLPs in a dose-dependent and PPxY-dependent manner. Moreover, results obtained with confocal and total internal reflection fluorescence microscopy suggested that Amot's role in actin organization and dynamics also contributes to promoting eVP40-mediated egress. In summary, these findings reveal a functional and competitive interplay between virus and host proteins involving the multifunctional PPxY-containing adaptor Amot, which regulates both the Hippo pathway and actin dynamics. We propose that our results have wide-ranging implications for understanding the biology and pathology of EBOV infections.
Asunto(s)
Ebolavirus/fisiología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Secuencias de Aminoácidos , Angiomotinas , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/transmisión , Fiebre Hemorrágica Ebola/virología , Vía de Señalización Hippo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Microscopía Confocal , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/metabolismo , Virión/fisiología , Liberación del VirusRESUMEN
Marburg virus (MARV) VP40 protein (mVP40) directs egress and spread of MARV, in part, by recruiting specific host WW domain-containing proteins via its conserved PPxY late (L) domain motif to facilitate efficient virus-cell separation. We reported previously that small-molecule compounds targeting the viral PPxY/host WW domain interaction inhibited VP40-mediated egress and spread. Here, we report on the antiviral potency of novel compound FC-10696, which emerged from extensive structure-activity relationship (SAR) of a previously described series of PPxY inhibitors. We show that FC-10696 inhibits egress of mVP40 virus-like particles (VLPs) and egress of authentic MARV from HeLa cells and primary human macrophages. Moreover, FC-10696 treated-mice displayed delayed onset of weight loss and clinical signs and significantly lower viral loads compared to controls, with 14% of animals surviving 21 days following a lethal MARV challenge. Thus, FC-10696 represents a first-in-class, host-oriented inhibitor effectively targeting late stages of the MARV life cycle.
Asunto(s)
Marburgvirus , Animales , Células HeLa , Humanos , Ratones , Liberación del VirusRESUMEN
Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Autofagia/fisiología , Ebolavirus/metabolismo , Fiebre Hemorrágica Ebola/transmisión , Enfermedad del Virus de Marburg/transmisión , Marburgvirus/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus/genética , Animales , Autofagia/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cricetinae , Ebolavirus/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte , Células HEK293 , Células HeLa , Fiebre Hemorrágica Ebola/patología , Fiebre Hemorrágica Ebola/virología , Humanos , Enfermedad del Virus de Marburg/patología , Enfermedad del Virus de Marburg/virología , Marburgvirus/genética , Ubiquitina-Proteína Ligasas Nedd4 , Prolina/análogos & derivados , Prolina/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006132.].
RESUMEN
Ebola virus (EBOV) is a member of the Filoviridae family and the cause of hemorrhagic fever outbreaks. The EBOV VP40 (eVP40) matrix protein is the main driving force for virion assembly and budding. Indeed, expression of eVP40 alone in mammalian cells results in the formation and budding of virus-like particles (VLPs) which mimic the budding process and morphology of authentic, infectious EBOV. To complete the budding process, eVP40 utilizes its PPXY L-domain motif to recruit a specific subset of host proteins containing one or more modular WW domains that then function to facilitate efficient production and release of eVP40 VLPs. In this report, we identified additional host WW-domain interactors by screening for potential interactions between mammalian proteins possessing one or more WW domains and WT or PPXY mutant peptides of eVP40. We identified the HECT family E3 ubiquitin ligase WWP1 and all four of its WW domains as strong interactors with the PPXY motif of eVP40. The eVP40-WWP1 interaction was confirmed by both peptide pulldown and coimmunoprecipitation assays, which also demonstrated that modular WW domain 1 of WWP1 was most critical for binding to eVP40. Importantly, the eVP40-WWP1 interaction was found to be biologically relevant for VLP budding since (i) small interfering RNA (siRNA) knockdown of endogenous WWP1 resulted in inhibition of eVP40 VLP egress, (ii) coexpression of WWP1 and eVP40 resulted in ubiquitination of eVP40 and a subsequent increase in eVP40 VLP egress, and (iii) an enzymatically inactive mutant of WWP1 (C890A) did not ubiquitinate eVP40 or enhance eVP40 VLP egress. Last, our data show that ubiquitination of eVP40 by WWP1 enhances egress of VLPs and concomitantly decreases cellular levels of higher-molecular-weight oligomers of eVP40. In sum, these findings contribute to our fundamental understanding of the functional interplay between host E3 ligases, ubiquitination, and regulation of EBOV VP40-mediated egress.IMPORTANCE Ebola virus (EBOV) is a high-priority, emerging human pathogen that can cause severe outbreaks of hemorrhagic fever with high mortality rates. As there are currently no approved vaccines or treatments for EBOV, a better understanding of the biology and functions of EBOV-host interactions that promote or inhibit viral budding is warranted. Here, we describe a physical and functional interaction between EBOV VP40 (eVP40) and WWP1, a host E3 ubiquitin ligase that ubiquitinates VP40 and regulates VLP egress. This viral PPXY-host WW domain-mediated interaction represents a potential new target for host-oriented inhibitors of EBOV egress.
Asunto(s)
Ebolavirus/fisiología , Interacciones Huésped-Patógeno , Nucleoproteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Liberación del Virus , Células HEK293 , Humanos , Nucleoproteínas/química , Nucleoproteínas/genética , ARN Interferente Pequeño , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/genética , Proteínas de la Matriz Viral/metabolismo , Virión/fisiología , Ensamble de VirusRESUMEN
UNLABELLED: Ebola virus (EBOV) and Marburg virus (MARV) belong to the Filoviridae family and can cause outbreaks of severe hemorrhagic fever, with high mortality rates in humans. The EBOV VP40 (eVP40) and MARV VP40 (mVP40) matrix proteins play a central role in virion assembly and egress, such that independent expression of VP40 leads to the production and egress of virus-like particles (VLPs) that accurately mimic the budding of infectious virus. Late (L) budding domains of eVP40 recruit host proteins (e.g., Tsg101, Nedd4, and Alix) that are important for efficient virus egress and spread. For example, the PPxY-type L domain of eVP40 and mVP40 recruits the host Nedd4 E3 ubiquitin ligase via its WW domains to facilitate budding. Here we sought to identify additional WW domain host interactors and demonstrate that the PPxY L domain motif of eVP40 interacts specifically with the WW domain of the host E3 ubiquitin ligase ITCH. ITCH, like Nedd4, is a member of the HECT class of E3 ubiquitin ligases, and the resultant physical and functional interaction with eVP40 facilitates VLP and virus budding. Identification of this novel eVP40 interactor highlights the functional interplay between cellular E3 ligases, ubiquitination, and regulation of VP40-mediated egress. IMPORTANCE: The unprecedented magnitude and scope of the recent 2014-2015 EBOV outbreak in West Africa and its emergence here in the United States and other countries underscore the critical need for a better understanding of the biology and pathogenesis of this emerging pathogen. We have identified a novel and functional EBOV VP40 interactor, ITCH, that regulates VP40-mediated egress. This virus-host interaction may represent a new target for our previously identified small-molecule inhibitors of virus egress.
Asunto(s)
Ebolavirus/fisiología , Interacciones Huésped-Patógeno , Nucleoproteínas/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas del Núcleo Viral/metabolismo , Liberación del Virus , Animales , Línea Celular , Humanos , Mapeo de Interacción de ProteínasRESUMEN
Hemorrhagic fever viruses, including the filoviruses (Ebola and Marburg) and arenaviruses (Lassa and Junín viruses), are serious human pathogens for which there are currently no FDA approved therapeutics or vaccines. Importantly, transmission of these viruses, and specifically late steps of budding, critically depend upon host cell machinery. Consequently, strategies which target these mechanisms represent potential targets for broad spectrum host oriented therapeutics. An important cellular signal implicated previously in EBOV budding is calcium. Indeed, host cell calcium signals are increasingly being recognized to play a role in steps of entry, replication, and transmission for a range of viruses, but if and how filoviruses and arenaviruses mobilize calcium and the precise stage of virus transmission regulated by calcium have not been defined. Here we demonstrate that expression of matrix proteins from both filoviruses and arenaviruses triggers an increase in host cytoplasmic Ca2+ concentration by a mechanism that requires host Orai1 channels. Furthermore, we demonstrate that Orai1 regulates both VLP and infectious filovirus and arenavirus production and spread. Notably, suppression of the protein that triggers Orai activation (Stromal Interaction Molecule 1, STIM1) and genetic inactivation or pharmacological blockade of Orai1 channels inhibits VLP and infectious virus egress. These findings are highly significant as they expand our understanding of host mechanisms that may broadly control enveloped RNA virus budding, and they establish Orai and STIM1 as novel targets for broad-spectrum host-oriented therapeutics to combat these emerging BSL-4 pathogens and potentially other enveloped RNA viruses that bud via similar mechanisms.
Asunto(s)
Arenavirus/fisiología , Filoviridae/fisiología , Liberación del Virus , Animales , Calcio/metabolismo , Canales de Calcio/fisiología , Células HEK293 , Células HeLa , Humanos , Proteína ORAI1 , Células Vero , Proteínas de la Matriz Viral/fisiología , Virión/fisiologíaRESUMEN
We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4.
Asunto(s)
Antivirales/farmacología , Inhibidores del Citocromo P-450 CYP3A/farmacología , Quinoxalinas/farmacología , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Proteínas de la Matriz Viral/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Citocromo P-450 CYP3A/metabolismo , Inhibidores del Citocromo P-450 CYP3A/síntesis química , Inhibidores del Citocromo P-450 CYP3A/química , Relación Dosis-Respuesta a Droga , Ebolavirus/química , Células HEK293 , Humanos , Marburgvirus/química , Ratones , Pruebas de Sensibilidad Microbiana , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Quinoxalinas/síntesis química , Quinoxalinas/química , Relación Estructura-ActividadRESUMEN
Ebola (EBOV) is an enveloped, negative-sense RNA virus belonging to the family Filoviridae that causes hemorrhagic fever syndromes with high-mortality rates. To date, there are no licensed vaccines or therapeutics to control EBOV infection and prevent transmission. Consequently, the need to better understand the mechanisms that regulate virus transmission is critical to developing countermeasures. The EBOV VP40 matrix protein plays a central role in late stages of virion assembly and egress, and independent expression of VP40 leads to the production of virus-like particles (VLPs) by a mechanism that accurately mimics budding of live virus. VP40 late (L) budding domains mediate efficient virus-cell separation by recruiting host ESCRT and ESCRT-associated proteins to complete the membrane fission process. L-domains consist of core consensus amino acid motifs including PPxY, P(T/S)AP, and YPx(n)L/I, and EBOV VP40 contains overlapping PPxY and PTAP motifs whose interactions with Nedd4 and Tsg101, respectively, have been characterized extensively. Here, we present data demonstrating for the first time that EBOV VP40 possesses a third L-domain YPx(n)L/I consensus motif that interacts with the ESCRT-III protein Alix. We show that the YPx(n)L/I motif mapping to amino acids 18-26 of EBOV VP40 interacts with the Alix Bro1-V fragment, and that siRNA knockdown of endogenous Alix expression inhibits EBOV VP40 VLP egress. Furthermore, overexpression of Alix Bro1-V rescues VLP production of the budding deficient EBOV VP40 double PTAP/PPEY L-domain deletion mutant to wild-type levels. Together, these findings demonstrate that EBOV VP40 recruits host Alix via a YPx(n)L/I motif that can function as an alternative L-domain to promote virus egress.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Ebolavirus/genética , Ebolavirus/metabolismo , Eliminación de Secuencia/genética , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus/genética , Secuencias de Aminoácidos/genética , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Células HEK293 , Fiebre Hemorrágica Ebola/metabolismo , Fiebre Hemorrágica Ebola/virología , Humanos , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Terciaria de Proteína/genéticaRESUMEN
UNLABELLED: There are currently no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics to prevent or treat Argentine hemorrhagic fever (AHF). The causative agent of AHF is Junin virus (JUNV); a New World arenavirus classified as a National Institute of Allergy and Infectious Disease/Centers for Disease Control and Prevention category A priority pathogen. The PTAP late (L) domain motif within JUNV Z protein facilitates virion egress and transmission by recruiting host Tsg101 and other ESCRT complex proteins to promote scission of the virus particle from the plasma membrane. Here, we describe a novel compound (compound 0013) that blocks the JUNV Z-Tsg101 interaction and inhibits budding of virus-like particles (VLPs) driven by ectopic expression of the Z protein and live-attenuated JUNV Candid-1 strain in cell culture. Since inhibition of the PTAP-Tsg101 interaction inhibits JUNV egress, compound 0013 serves as a prototype therapeutic that could reduce virus dissemination and disease progression in infected individuals. Moreover, since PTAP l-domain-mediated Tsg101 recruitment is utilized by other RNA virus pathogens (e.g., Ebola virus and HIV-1), PTAP inhibitors such as compound 0013 have the potential to function as potent broad-spectrum, host-oriented antiviral drugs. IMPORTANCE: There are currently no FDA-approved vaccines or therapeutics to prevent or treat Argentine hemorrhagic fever (AHF). The causative agent of AHF is Junin virus (JUNV); a New World arenavirus classified as an NIAID/CDC category A priority pathogen. Here, we describe a prototype therapeutic that blocks budding of JUNV and has the potential to function as a broad-spectrum antiviral drug.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Interacciones Huésped-Patógeno , Virus Junin/inmunología , Virus Junin/fisiología , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Liberación del Virus , Animales , Línea Celular , Humanos , Unión ProteicaRESUMEN
UNLABELLED: Budding of filoviruses, arenaviruses, and rhabdoviruses is facilitated by subversion of host proteins, such as Nedd4 E3 ubiquitin ligase, by viral PPxY late (L) budding domains expressed within the matrix proteins of these RNA viruses. As L domains are important for budding and are highly conserved in a wide array of RNA viruses, they represent potential broad-spectrum targets for the development of antiviral drugs. To identify potential competitive blockers, we used the known Nedd4 WW domain-PPxY interaction interface as the basis of an in silico screen. Using PPxY-dependent budding of Marburg (MARV) VP40 virus-like particles (VLPs) as our model system, we identified small-molecule hit 1 that inhibited Nedd4-PPxY interaction and PPxY-dependent budding. This lead candidate was subsequently improved with additional structure-activity relationship (SAR) analog testing which enhanced antibudding activity into the nanomolar range. Current lead compounds 4 and 5 exhibit on-target effects by specifically blocking the MARV VP40 PPxY-host Nedd4 interaction and subsequent PPxY-dependent egress of MARV VP40 VLPs. In addition, lead compounds 4 and 5 exhibited antibudding activity against Ebola and Lassa fever VLPs, as well as vesicular stomatitis and rabies viruses (VSV and RABV, respectively). These data provide target validation and suggest that inhibition of the PPxY-Nedd4 interaction can serve as the basis for the development of a novel class of broad-spectrum, host-oriented antivirals targeting viruses that depend on a functional PPxY L domain for efficient egress. IMPORTANCE: There is an urgent and unmet need for the development of safe and effective therapeutics against biodefense and high-priority pathogens, including filoviruses (Ebola and Marburg) and arenaviruses (e.g., Lassa and Junin) which cause severe hemorrhagic fever syndromes with high mortality rates. We along with others have established that efficient budding of filoviruses, arenaviruses, and other viruses is critically dependent on the subversion of host proteins. As disruption of virus budding would prevent virus dissemination, identification of small-molecule compounds that block these critical viral-host interactions should effectively block disease progression and transmission. Our findings provide validation for targeting these virus-host interactions as we have identified lead inhibitors with broad-spectrum antiviral activity. In addition, such inhibitors might prove useful for newly emerging RNA viruses for which no therapeutics would be available.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Preparaciones Farmacéuticas/metabolismo , Unión Proteica/efectos de los fármacos , Infecciones por Virus ARN/tratamiento farmacológico , Virus ARN/fisiología , Bibliotecas de Moléculas Pequeñas , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus/efectos de los fármacos , Western Blotting , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Prueba de Complementación Genética , Humanos , Ubiquitina-Proteína Ligasas Nedd4 , Infecciones por Virus ARN/virología , Virus ARN/efectos de los fármacos , ARN Viral/genética , Relación Estructura-Actividad , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Proteínas de la Matriz Viral/antagonistas & inhibidores , Virión/efectos de los fármacos , Virión/fisiologíaRESUMEN
We have identified host IQGAP1 as an interacting partner for Ebola virus (EBOV) VP40, and its expression is required for EBOV VP40 virus-like particle (VLP) budding. IQGAP1 is involved in actin cytoskeletal remodeling during cell migration and formation of filopodia. The physical interaction and the functional requirement for IQGAP1 in EBOV VP40 VLP egress link virus budding to the cytoskeletal remodeling machinery. Consequently, this interaction represents a novel target for development of therapeutics to block budding and transmission of filoviruses.
Asunto(s)
Ebolavirus/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de la Matriz Viral/metabolismo , Liberación del Virus/fisiología , Proteínas Activadoras de ras GTPasa/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Componentes del Gen , Células HEK293 , Humanos , Microscopía Fluorescente , ARN Interferente Pequeño/genética , Transfección , Virión/metabolismo , Liberación del Virus/genética , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.
Asunto(s)
Antivirales/farmacología , Virosis/tratamiento farmacológico , Replicación Viral/efectos de los fármacos , Animales , Antibacterianos/química , Antibacterianos/farmacología , Antivirales/química , Línea Celular , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Células Cultivadas , Colestanoles/química , Colestanoles/farmacología , Cricetinae , Femenino , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/crecimiento & desarrollo , Virus de la Hepatitis Delta/efectos de los fármacos , Virus de la Hepatitis Delta/crecimiento & desarrollo , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Masculino , Mesocricetus , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Muromegalovirus/efectos de los fármacos , Muromegalovirus/crecimiento & desarrollo , Dispersión del Ángulo Pequeño , Virosis/virología , Difracción de Rayos X , Proteína de Unión al GTP rac1/químicaRESUMEN
Helicobacter pylori infection, a worldwide health issue, is typically treated with standard antibiotic therapies. However, these treatments often face resistance and non-compliance due to side effects. In this umbrella review, we aimed to comprehensively assess the impact of probiotics supplementation in different preparations on Helicobacter pylori standard treatment. We searched PubMed, Embase and Cochrane Central Register of Controlled Trials in the Cochrane Library from inception to June 1, 2023, to identify systematic reviews with meta-analyses that focused on eradication rates, total side effects and other outcomes of interest. The most comprehensive meta-analysis was selected for data extraction. AMSTAR 2 was used to assess quality of meta-analyses. Overall, 28 unique meta-analyses based on 534 RCTs were included. The results suggests that probiotics supplementation with pooled probiotic strains was significantly associated with improved eradication rates (RR 1.10, 95% CI 1.06-1.14) and reduced risk of total side effects (RR 0.54, 95% CI 0.42-0.70) compared with standard therapy alone. Single-strained or multi-strained preparation of probiotics supplementation showed similar results. Despite Bifidobacterium spp. showing the highest potential for eradication, the study quality was critically low for most meta-analyses, necessitating further high-quality research to explore the optimal probiotic strains or their combinations for Helicobacter pylori treatment.aq_start?>Kindly check and confirm the edit made in article title.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Probióticos , Revisiones Sistemáticas como Asunto , Probióticos/uso terapéutico , Helicobacter pylori/efectos de los fármacos , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/terapia , Infecciones por Helicobacter/microbiología , Humanos , Metaanálisis como Asunto , Suplementos Dietéticos , Antibacterianos/uso terapéutico , Resultado del TratamientoRESUMEN
OBJECTIVE: Pemphigus vulgaris (PV) and bullous pemphigoid (BP) are two prevalent bullous diseases. Previous studies found that the antibodies of BP could be expressed in the intestinal epithelium and BP was tightly related to inflammatory bowel disease. Therefore, gut microbiota might also play an important role in bullous disease. However, the specific relationship between gut microbiota and bullous diseases remains unknown. Our study aimed to investigate the potential role of gut microbiota in the development and progression of different bullous diseases. METHODS: We conducted a prospective and observational cohort study at Peking Union Medical College Hospital. Untreated BP and PV patients were recruited, along with healthy controls (HC) who were spouses or caregivers of these patients. Fecal samples were collected, followed by 16S rRNA gene sequencing. Bioinformatics analyses were performed to assess the composition and function of gut microbiota. RESULTS: A total of 38 HC, 32 BP, and 19 PV patients were enrolled in this study. Compared to HC, BP, and PV exhibited a distinct gut microbiota composition, especially BP. The gut microbiota changes were mainly observed in the phylum Bacteroidetes, Firmicutes, and Proteobacteria. The ratio of Faecalibacterium to Escherichia-Shigella (F/E ratio) had a considerable predictive value (AUC: 0.705) for recognizing BP from PV. The levels of Faecalibacterium and Enterobacter were correlated to the anti-BP 180 and anti-desmoglein 3. Microbial functional prediction revealed elevated activity in pathways related to gut microbiota translocation significantly increased in BP patients, indicating a potential pathogenetic role in BP. CONCLUSIONS: Our study suggests that the composition of gut microbiota is specific in different bullous diseases and the role of gut microbiota differs. Gut microbiota could help distinguish BP and PV, and might play a role in the pathogenesis of different bullous diseases.
Asunto(s)
Microbioma Gastrointestinal , Penfigoide Ampolloso , Pénfigo , Humanos , Penfigoide Ampolloso/patología , Estudios Prospectivos , ARN Ribosómico 16S , DisbiosisRESUMEN
Background: Acute respiratory distress syndrome (ARDS) is the most common cause of organ failure in acute pancreatitis (AP) patients, which associated with high mortality. Specific changes in the gut microbiota have been shown to influence progression of acute pancreatitis. We aimed to determine whether early alterations in the gut microbiota is related to and could predict ARDS occurrence in AP patients. Methods: In this study, we performed 16S rRNA sequencing analysis in 65 AP patients and 20 healthy volunteers. The AP patients were further divided into two groups: 26 AP-ARDS patients and 39 AP-nonARDS patients based on ARDS occurrence during hospitalization. Results: Our results showed that the AP-ARDS patients exhibited specific changes in gut microbiota composition and function as compared to subjects of AP-nonARDS group. Higher abundances of Proteobacteria phylum, Enterobacteriaceae family, Escherichia-Shigella genus, and Klebsiella pneumoniae, but lower abundances of Bifidobacterium genus were found in AP-ARDS group compared with AP-nonARDS groups. Random forest modelling analysis revealed that the Escherichia-shigella genus was effective to distinguish AP-ARDS from AP-nonARDS, which could predict ARDS occurrence in AP patients. Conclusions: Our study revealed that alterations of gut microbiota in AP patients on admission were associated with ARDS occurrence after hospitalization, indicating a potential predictive and pathogenic role of gut microbiota in the development of ARDS in AP patients.