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Reticulon-3 Modulates the Specific Incorporation of Replication Competent Hepatitis C Virus Molecules for Release Via Exosomes

Li, Jingjing, Abosmaha, e, Labonté, Patrick ORCID: https://orcid.org/0000-0001-7262-3125 et Bukong, Terence Ndonyi (2019). Reticulon-3 Modulates the Specific Incorporation of Replication Competent Hepatitis C Virus Molecules for Release Via Exosomes In: Liver Meeting of the American Association for the Study of Liver Diseases (AASLD), 8-12 November 2019, Boston, Ma.

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Résumé

Background: Exosomes play an important role in mediating immunologic escape, treatment resistance and disease persistence of almost all hepatic viral infections. Central to this pathomechanistic process is the specific incorporation of host and viral molecules inside infectious exosomes. The precise molecular mechanisms directing the selective cargo sorting and loading inside infectious exosomes remains elusive. Aim: To determine the mechanistic role of Reticulon 3 (RTN3) in the selective cargo loading of specific host and replication competent Hepatitis C Virus (HCV) material for release inside exosomes. Methods: We took advantage of the Huh7 cells – JFH1 HCV infection and HCV Full-Length (FL) replicon systems. Additionally, we made use of human liver and serum exosome samples from healthy and HCV infected patients. Our experimental analysis made use of molecular biology and immunology techniques including confocal microscopy, gene modulation, electron microscopy, NanoSight, quantitative RT-qPCR and western blot analysis. Results: We reveal that HCV infection of Huh7 cells in the context of JFH1 or HCV-FL replicon cells was associated with increased cellular expression of both long and short isoforms of RTN3 (RTN3L & RTN3S). Accordingly, increased RTN3 expression was observed in HCV infected patient liver samples compared to healthy control subjects. Confocal microscopy of HCV infected hepatocytes revealed that RTN3S&L co-localised with dsHCV RNA suggesting possible interaction. Characterization revealed that control and infectious exosomes contained TSG101 but no Calnexin, suggesting no microsomes contamination of our exosome preparations. Infectious exosomes were specifically enriched with RTN3L, dsHCV RNA, Ago2, miR122, and HSP90. RTN3S was not detected in control or infectious exosomes. Transient knockdown (KD) of RTN3 and overexpression (OE) of wild type, C- and N-terminal deletion mutants of RTN3S&L in HCV-JFH1 infected Huh7 cells did not affect HCV NS3 protein and HCV RNA expressions suggesting that RTN3 did not impact HCV replication. RTN3 KD in hepatocytes (Huh7 and FL Replicon cells) significantly decreased, while RTN3 OE significantly increased the number of cell-released exosomes. Strikingly, cellular OE of C-terminal deleted RTN3S mutant in HCV infected hepatocytes was associated with a significant increase in RTN3L. These observations suggest possible compensatory OE of RTN3L following RTN3S c-terminal deletion mutant OE. Ultimately, co-culture experiments revealed that exosomes from HCV infected RTN3 KD hepatocytes were significantly less infectious. Conclusion: This study revealed RTN3 as a novel regulator and therapeutic target that can be exploited to prevent the cellular release of infectious exosomes.

Type de document: Document issu d'une conférence ou d'un atelier
Informations complémentaires: Affiche scientifique: 1659 Hepatology 70 (s1):999A-999A
Mots-clés libres: -
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 23 juill. 2021 17:11
Dernière modification: 15 févr. 2022 18:21
URI: https://espace.inrs.ca/id/eprint/11729

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