Hepatitis E virus RNA-dependent RNA polymerase is involved in RNA replication and infectious particle production.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and is known as the viral replicase. The RNA-dependent RNA polymerase (RDRP) domain is responsible for the synthesis of viral RNA. Here, we identified a highly conserved α-helix located in the RDRP thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RDRP palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release. Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RDRP with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RDRP thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production

Fluid shear stress modulation of hepatocyte like cell function

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype

Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion.

Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle

Liver cell therapy: is this the end of the beginning?

The prevalence of liver diseases is increasing globally. Orthotopic liver transplantation is widely used to treat liver disease upon organ failure. The complexity of this procedure and finite numbers of healthy organ donors have prompted research into alternative therapeutic options to treat liver disease. This includes the transplantation of liver cells to promote regeneration. While successful, the routine supply of good quality human liver cells is limited. Therefore, renewable and scalable sources of these cells are sought. Liver progenitor and pluripotent stem cells offer potential cell sources that could be used clinically. This review discusses recent approaches in liver cell transplantation and requirements to improve the process, with the ultimate goal being efficient organ regeneration. We also discuss the potential off-target effects of cell-based therapies, and the advantages and drawbacks of current pre-clinical animal models used to study organ senescence, repopulation and regeneration

Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle

Effect of Sheep’s Whey Edible Coatings with a Bioprotective Culture, Kombucha Tea or Oregano Essential Oil on Cheese Characteristics

Films and coatings based on biopolymers have been extensively studied in recent years since they have less impact on the environment, can be obtained from renewable sources, have good coating and film-forming capacity, are biodegradable and can have interesting nutritional properties. In the present study, sheep’s cheese whey powder (SCWP) was used to produce edible cheese coatings. Six types of cheese samples were produced: without coating (CON); treated with natamycin (NAT); with SCWP coating without antimicrobials (WCO); with SCWP coating with a commercial bioprotective culture (WFQ); with SCWP coating with kombucha tea (WKO); and with SCWP coating with oregano essential oil (WEO). At the end of the ripening period, all the cheeses were classified as full-fat and semihard, according to the Portuguese standard. The higher hardness and adhesiveness values of samples CON, WFQ and WKO were in line with the lower moisture in defatted cheese observed in these samples, indicating that future work should address the improvement of water vapor barrier properties of the whey-based coating. The samples treated with natamycin and with oregano essential oil presented significantly lower values for hardness. Differences were also observed on titratable acidity and aw, both between samples and because of ripening time. The color parameters of cheese samples also presented differences, chiefly in the rind, but the highest differences observed resulted from ripening time rather than between samples. In all cases, the counts of lactobacilli and lactococci surpassed log 7 CFU/g by the end of ripening. Regarding yeast and mold counts, the samples CON and WCO presented the highest values by the end of the ripening period (>log 4 CFU/g), while sample NAT presented the lowest value (ca. log 3 CFU/g). Samples WFQ, WKO and WEO presented values which were ca. 0.5 log cycles lower than samples CON and WCO. Hence, the use of SCWP alongside bioprotective culture, kombucha tea or oregano essential oil had a positive impact in the reduction of mold counts on cheese surfaces. Future work should also evaluate the joint use of different antimicrobials