Boulon, Richard; Blanchet, Matthieu; Vaillant, Andrew et Labonté, Patrick . The Hsp40 chaperone DnaJB12 is involved in the morphogenesis of HBV spherical subviral particles and is selectively targeted by nucleic acid polymers In: 19th Annual International Congress of the International Liver Transplantation Society, 8-12 July 2020, Sydnay, Australie.
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Background:Nucleic acid polymers (NAPs) inhibit the assembly and secretion of HBV spherical subviral particles (SVP) without affecting the secretion of HBeAg or Daneparticles. Given that > 99.99% of circulating HBsAg is derived from spherical SVP, the importance of HBsAg loss in achieving functional cure of HBV and the high rates of HBsAg loss and functional cure uniquely accompanying NAP-based therapy, the host target(s) of NAPs has been a topic of great interest. Recent validation of thein vivoand clinical effects of NAP effects in HepG2.2.15 cells (Blanchetet al., Antiviral Res. 2019;164:97-105, Boulonet al., Antiviral Res. 2020;180:104853) identifies a suitable model system for NAP target identification.Methods:A differential-interactome screen of HepG2.2.15 lysate used biotinylated NAPs which bracket the size and phosphorothioation (PS) dependent structure activity relationship of NAPs (Blanchetet al.,ibid). These NAPs included the clinically active 40mer PS REP 2139 and its inactive analogs: the 40mer phosphodiester REP 2147 and the short PS(20mer) REP 2179.MS/MS analysis (three experiments per NAP) identified NAP-bound proteins. DNA / RNA binding proteins or proteins with interaction selectivity ratio < 2 were excluded. Selected candidates had the greatest significant (p < 0.05) selective interaction ratio between REP 2139 / REP 2147 (PS-dependent) and REP 2139 / REP 2179 (size-dependent). Candidateswere validated by shRNA-mediated knockdown effects on HBsAg and HBeAg secretion. Reproducibility (n=3) of validation experiments was recently confirmed.Results:No interactions with viral proteins were detected. Knockdown of candidate targets in HepG2.2.15 cells revealed two targets involved in HBsAg secretion: the Hsp40 chaperone DNAJB12 and casein kinase 1 isoform delta (CSNK1D), involved in retrograde vesicle transport. Knockdown of DNAJB12 resulted in the strongest inhibition of HBsAg secretion with no effect on HBeAg. Knockdown of CSNK1D induced milder inhibition of both HBsAg and HBeAg secretion (inconsistent with NAP activityin vitroand in humans). No selective interactions were observed with other Hsp40 family DNAJ members, including DNAJB1 and DNAJB4 involved in regulating preS1 membrane topology during Dane particle morphogenesis.Conclusion:The selective inhibition of spherical SVP assembly by NAPs is a result of the inhibition of DNAJB12-mediated chaperone functions required for spherical SVPassembly. NAP interaction with CSNK1D does not appear to be physiological but may reflect an interaction which can occurin vitrounder experimental conditions which do not mimic normal intracellular NAP trafficking.
| Type de document: | Document issu d'une conférence ou d'un atelier |
|---|---|
| Informations complémentaires: | Présentation orale no LP 42 |
| Mots-clés libres: | - |
| Centre: | Centre INRS-Institut Armand Frappier |
| Date de dépôt: | 14 juill. 2021 15:53 |
| Dernière modification: | 14 juill. 2021 15:53 |
| URI: | https://espace.inrs.ca/id/eprint/11826 |
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