Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.
The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded protease...
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Public Library of Science (PLoS)
2023-07-01
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| Series: | PLoS Pathogens |
| Online Access: | https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1011529&type=printable |
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| author | Danielle M Pierce Frazer J T Buchanan Fraser L Macrae Jake T Mills Abigail Cox Khadijah M Abualsaoud Joseph C Ward Robert A S Ariëns Mark Harris Nicola J Stonehouse Morgan R Herod |
| author_facet | Danielle M Pierce Frazer J T Buchanan Fraser L Macrae Jake T Mills Abigail Cox Khadijah M Abualsaoud Joseph C Ward Robert A S Ariëns Mark Harris Nicola J Stonehouse Morgan R Herod |
| author_sort | Danielle M Pierce |
| collection | DOAJ |
| description | The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing. |
| format | Article |
| id | doaj-art-8056a19c68d34c99a2ca5b5329f1d0ff |
| institution | OA Journals |
| issn | 1553-7366 1553-7374 |
| language | English |
| publishDate | 2023-07-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj-art-8056a19c68d34c99a2ca5b5329f1d0ff2025-08-20T02:31:38ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742023-07-01197e101152910.1371/journal.ppat.1011529Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.Danielle M PierceFrazer J T BuchananFraser L MacraeJake T MillsAbigail CoxKhadijah M AbualsaoudJoseph C WardRobert A S AriënsMark HarrisNicola J StonehouseMorgan R HerodThe genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1011529&type=printable |
| spellingShingle | Danielle M Pierce Frazer J T Buchanan Fraser L Macrae Jake T Mills Abigail Cox Khadijah M Abualsaoud Joseph C Ward Robert A S Ariëns Mark Harris Nicola J Stonehouse Morgan R Herod Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication. PLoS Pathogens |
| title | Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication. |
| title_full | Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication. |
| title_fullStr | Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication. |
| title_full_unstemmed | Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication. |
| title_short | Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication. |
| title_sort | thrombin cleavage of the hepatitis e virus polyprotein at multiple conserved locations is required for genome replication |
| url | https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1011529&type=printable |
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