The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummary
Background: A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. Methods: Subcellular localization of pORF1 and its subdomain...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-01-01
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| Series: | Cellular and Molecular Gastroenterology and Hepatology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352345X24000018 |
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| author | Mirco Glitscher Inga Mareike Spannaus Fabiane Behr Robin Oliver Murra Kathrin Woytinek Daniela Bender Eberhard Hildt |
| author_facet | Mirco Glitscher Inga Mareike Spannaus Fabiane Behr Robin Oliver Murra Kathrin Woytinek Daniela Bender Eberhard Hildt |
| author_sort | Mirco Glitscher |
| collection | DOAJ |
| description | Background: A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. Methods: Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase–polymerase chain reaction to analyze separated particles in more detail. Results: We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein’s 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3. Conclusions: Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1. |
| first_indexed | 2024-03-07T23:06:30Z |
| format | Article |
| id | doaj.art-d7e61af89377460ca741b230e6b20336 |
| institution | Directory Open Access Journal |
| issn | 2352-345X |
| language | English |
| last_indexed | 2024-04-24T23:24:17Z |
| publishDate | 2024-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cellular and Molecular Gastroenterology and Hepatology |
| spelling | doaj.art-d7e61af89377460ca741b230e6b203362024-03-16T05:08:35ZengElsevierCellular and Molecular Gastroenterology and Hepatology2352-345X2024-01-01174589605The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummaryMirco Glitscher0Inga Mareike Spannaus1Fabiane Behr2Robin Oliver Murra3Kathrin Woytinek4Daniela Bender5Eberhard Hildt6Department of Virology, Paul-Ehrlich-Institute, Langen, GermanyDepartment of Virology, Paul-Ehrlich-Institute, Langen, GermanyDepartment of Virology, Paul-Ehrlich-Institute, Langen, GermanyDepartment of Virology, Paul-Ehrlich-Institute, Langen, GermanyDepartment of Virology, Paul-Ehrlich-Institute, Langen, GermanyDepartment of Virology, Paul-Ehrlich-Institute, Langen, GermanyCorrespondence Address correspondence to: Prof Dr Eberhard Hildt, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany.; Department of Virology, Paul-Ehrlich-Institute, Langen, GermanyBackground: A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. Methods: Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase–polymerase chain reaction to analyze separated particles in more detail. Results: We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein’s 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3. Conclusions: Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1.http://www.sciencedirect.com/science/article/pii/S2352345X24000018PCPExosomesViral ReplicationViral Spread |
| spellingShingle | Mirco Glitscher Inga Mareike Spannaus Fabiane Behr Robin Oliver Murra Kathrin Woytinek Daniela Bender Eberhard Hildt The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummary Cellular and Molecular Gastroenterology and Hepatology PCP Exosomes Viral Replication Viral Spread |
| title | The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummary |
| title_full | The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummary |
| title_fullStr | The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummary |
| title_full_unstemmed | The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummary |
| title_short | The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal ReleaseSummary |
| title_sort | protease domain in hev porf1 mediates the replicase s localization to multivesicular bodies and its exosomal releasesummary |
| topic | PCP Exosomes Viral Replication Viral Spread |
| url | http://www.sciencedirect.com/science/article/pii/S2352345X24000018 |
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