RESUMO
An endolichenic strain of the Ascomycetaceous Xylaria hypoxylon, cultivated alone or in coculture with another endolichenic fungus Dendrothyrium variisporum, produced seven new bioactive eremophilane sesquiterpenes eremoxylarins D-J (1-7). The isolated compounds disclosed a high similarity with the eremophilane core of the bioactive integric acid, and structures were elucidated by 1D and 2D NMR spectra and electronic circular dichroism (ECD) analyses. Eremoxylarins D, F, G, and I showed a selective activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus with minimum inhibitory concentration (MIC) values between 0.39 and 12.5 µg/mL. Eremoxylarin I, the most antibacterial active sesquiterpene, was also active against HCoV-229E at a concentration nontoxic to the hepatoma Huh-7 cell line with an 50% inhibitory concentration (IC50) of 18.1 µM and a 50% cytotoxic concentration (CC50) of 46.6 µM.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Sesquiterpenos , Sesquiterpenos Policíclicos , Sesquiterpenos/química , Antibacterianos/química , Estrutura MolecularRESUMO
Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically transmitted viral hepatitis worldwide. Ribavirin (RBV) is currently the only treatment option for many patients; however, cases of treatment failures or posttreatment relapses have been frequently reported. RBV therapy was shown to be associated with an increase in HEV genome heterogeneity and the emergence of distinct HEV variants. In this study, we analyzed the impact of eight patient-derived open reading frame 2 (ORF2) single-nucleotide variants (SNVs), which occurred under RBV treatment, on the replication cycle and pathogenesis of HEV. The parental HEV strain and seven ORF2 variants showed comparable levels of RNA replication in human hepatoma cells and primary human hepatocytes. However, a P79S ORF2 variant demonstrated reduced RNA copy numbers released in the supernatant and an impairment in the production of infectious particles. Biophysical and biochemical characterization revealed that this SNV caused defective, smaller HEV particles with a loss of infectiousness. Furthermore, the P79S variant displayed an altered subcellular distribution of the ORF2 protein and was able to interfere with antibody-mediated neutralization of HEV in a competition assay. In conclusion, an SNV in the HEV ORF2 could be identified that resulted in altered virus particles that were noninfectious in vitro and in vivo, but could potentially serve as immune decoys. These findings provide insights in understanding the biology of circulating HEV variants and may guide development of personalized antiviral strategies in the future.
Assuntos
Vírus da Hepatite E , Ribavirina , Proteínas Virais , Linhagem Celular Tumoral , Vírus da Hepatite E/genética , Vírus da Hepatite E/fisiologia , Hepatócitos/virologia , Humanos , Recidiva Local de Neoplasia/genética , Nucleotídeos , RNA Viral , Ribavirina/farmacologia , Proteínas Virais/genética , Replicação ViralRESUMO
Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing three open reading frames (ORFs). ORF1 encodes the ORF1 non-structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all three systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope ®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.