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Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein
Dagmara Szkolnicka1, Angela Pollán1, Nathalie Da Silva1
1Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland.
Journal of Virology
|July 19, 2019
Summary
Researchers developed tagged Hepatitis E virus (HEV) genomes to study its life cycle. These tools enable tracking HEV infection and identifying replication sites, advancing understanding of this common cause of hepatitis.
Area of Science:
- Virology
- Molecular Biology
- Hepatitis Research
Background:
- Hepatitis E virus (HEV) is a leading cause of acute hepatitis globally.
- Understanding HEV's molecular virology and pathogenesis is hindered by a lack of functional tools.
- Limited sensitivity of current methods impedes the study of HEV replicase and ORF1 protein expression.
Purpose of the Study:
- To develop novel tagged HEV genomes for investigating the viral life cycle.
- To create functional tools for studying HEV replication and pathogenesis.
- To identify HEV RNA replication sites and characterize replication complexes.
Main Methods:
- Development of tagged HEV genomes using transposon-based random insertion into ORF1.
- Selection of viable insertions in a hepatoblastoma cell line.
- Utilizing HA-tagged and NanoLuc-tagged HEV genomes for infection monitoring and localization studies.
- Employing fluorescence in situ hybridization and immunofluorescence for detecting viral RNA and ORF1 protein.
Main Results:
- Functional insertion sites in HEV ORF1 were identified.
- HA-tagged and NanoLuc-tagged HEV genomes were fully functional, producing infectious virus.
- NanoLuc enabled quantitative monitoring of HEV infection and replication.
- Putative HEV replication complexes were localized in cytoplasmic dot-like structures.
Conclusions:
- Tagged HEV genomes are effective novel tools for studying the HEV life cycle.
- These tools facilitate further investigation into the structure and composition of the HEV replication complex.
- The developed tools enhance understanding of HEV pathogenesis and may aid in developing therapeutic strategies.
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