Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein
Background and Aims: Hepatitis E virus (HEV) infection is a leading cause of acute hepatitis worldwide. Understanding of the mechanisms underlying productive HEV infection remains incomplete and would benefit from technological advances improving current model systems. Methods: We exploited transpos...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | JHEP Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2589555924002970 |
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| author | Maliki Ankavay Nathalie Da Silva Angela Pollán Noémie Oechslin Katja Dinkelborg Patrick Behrendt Darius Moradpour Jérôme Gouttenoire |
| author_facet | Maliki Ankavay Nathalie Da Silva Angela Pollán Noémie Oechslin Katja Dinkelborg Patrick Behrendt Darius Moradpour Jérôme Gouttenoire |
| author_sort | Maliki Ankavay |
| collection | DOAJ |
| description | Background and Aims: Hepatitis E virus (HEV) infection is a leading cause of acute hepatitis worldwide. Understanding of the mechanisms underlying productive HEV infection remains incomplete and would benefit from technological advances improving current model systems. Methods: We exploited transposon-mediated random insertion and selection of viable clones to identify sites in the HEV open reading frame 2 (ORF2) protein, corresponding to the viral capsid, allowing for the insertion of reporter sequences in a functional context. Results: Short sequence insertions (5 amino acids) were tolerated at four distinct sites in the C-terminal region of the ORF2 protein, without significantly affecting viral capsid expression and subcellular localization as well as virus production. Full-length HEV genomes harboring larger sequence insertions such as an HA epitope tag, a highly sensitive miniaturized luciferase reporter (HiBiT) or a split GFP at these sites conserved their ability to produce infectious virus, with about a 1-log decrease in viral titers. Findings were confirmed in two different HEV genotype 3 clones. In addition, we demonstrate that HiBiT-tagged HEV, offering rapid and several-log amplitude detection, can be used for the evaluation of antiviral drugs and neutralizing antibodies. Conclusions: We describe a convenient, quantitative and potentially scalable system for the monitoring of HEV infection and replication in tissue culture. Impact and implications:: Hepatitis E virus infection is one of the most frequent causes of acute hepatitis and jaundice worldwide. As treatment options are limited and a vaccine is not universally available, the development of molecular tools to facilitate the identification of new therapeutic strategies is crucial. Based on a screening approach to identify viable insertion sites in the viral genome, we describe a versatile system for preparing recombinant viruses harboring split-reporter tags, i.e. luciferase and GFP. Proof-of-concept experiments revealed that convenient and quantitative monitoring of viral infection and replication is possible with this system, allowing for the evaluation of antiviral drugs and neutralizing antibodies. |
| format | Article |
| id | doaj-art-f99ca0b6aeba4df1ba220481dc47a93a |
| institution | Kabale University |
| issn | 2589-5559 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | JHEP Reports |
| spelling | doaj-art-f99ca0b6aeba4df1ba220481dc47a93a2025-02-04T04:10:32ZengElsevierJHEP Reports2589-55592025-03-0173101293Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 proteinMaliki Ankavay0Nathalie Da Silva1Angela Pollán2Noémie Oechslin3Katja Dinkelborg4Patrick Behrendt5Darius Moradpour6Jérôme Gouttenoire7Division of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandDivision of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandDivision of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandDivision of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandDepartment of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School and Institute for Experimental Virology, Twincore Centre for Experimental and Clinical Infection Research, Hannover, GermanyDepartment of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School and Institute for Experimental Virology, Twincore Centre for Experimental and Clinical Infection Research, Hannover, GermanyDivision of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, SwitzerlandDivision of Gastroenterology and Hepatology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; Corresponding author. Address: Division of Gastroenterology and Hepatology, Lausanne University Hospital (CHUV), Rue du Bugnon 48, CH-1011 Lausanne, Switzerland.Background and Aims: Hepatitis E virus (HEV) infection is a leading cause of acute hepatitis worldwide. Understanding of the mechanisms underlying productive HEV infection remains incomplete and would benefit from technological advances improving current model systems. Methods: We exploited transposon-mediated random insertion and selection of viable clones to identify sites in the HEV open reading frame 2 (ORF2) protein, corresponding to the viral capsid, allowing for the insertion of reporter sequences in a functional context. Results: Short sequence insertions (5 amino acids) were tolerated at four distinct sites in the C-terminal region of the ORF2 protein, without significantly affecting viral capsid expression and subcellular localization as well as virus production. Full-length HEV genomes harboring larger sequence insertions such as an HA epitope tag, a highly sensitive miniaturized luciferase reporter (HiBiT) or a split GFP at these sites conserved their ability to produce infectious virus, with about a 1-log decrease in viral titers. Findings were confirmed in two different HEV genotype 3 clones. In addition, we demonstrate that HiBiT-tagged HEV, offering rapid and several-log amplitude detection, can be used for the evaluation of antiviral drugs and neutralizing antibodies. Conclusions: We describe a convenient, quantitative and potentially scalable system for the monitoring of HEV infection and replication in tissue culture. Impact and implications:: Hepatitis E virus infection is one of the most frequent causes of acute hepatitis and jaundice worldwide. As treatment options are limited and a vaccine is not universally available, the development of molecular tools to facilitate the identification of new therapeutic strategies is crucial. Based on a screening approach to identify viable insertion sites in the viral genome, we describe a versatile system for preparing recombinant viruses harboring split-reporter tags, i.e. luciferase and GFP. Proof-of-concept experiments revealed that convenient and quantitative monitoring of viral infection and replication is possible with this system, allowing for the evaluation of antiviral drugs and neutralizing antibodies.http://www.sciencedirect.com/science/article/pii/S2589555924002970HEVHiBiTORF2 proteinrandom insertionsplit-luciferasetransposon |
| spellingShingle | Maliki Ankavay Nathalie Da Silva Angela Pollán Noémie Oechslin Katja Dinkelborg Patrick Behrendt Darius Moradpour Jérôme Gouttenoire Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein JHEP Reports HEV HiBiT ORF2 protein random insertion split-luciferase transposon |
| title | Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein |
| title_full | Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein |
| title_fullStr | Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein |
| title_full_unstemmed | Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein |
| title_short | Monitoring of hepatitis E virus infection and replication by functional tagging of the ORF2 protein |
| title_sort | monitoring of hepatitis e virus infection and replication by functional tagging of the orf2 protein |
| topic | HEV HiBiT ORF2 protein random insertion split-luciferase transposon |
| url | http://www.sciencedirect.com/science/article/pii/S2589555924002970 |
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