Elucidating new roles of ubiquitin ligases in the HBV life cycle

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F25%3A00643139" target="_blank" >RIV/61388963:_____/25:00643139 - isvavai.cz</a>

Result on the web

<a href="http://www.ccsss.cz/index.php/ccsss/issue/view/52/92" target="_blank" >http://www.ccsss.cz/index.php/ccsss/issue/view/52/92</a>

DOI - Digital Object Identifier

—

Result language

angličtina

Original language name

Elucidating new roles of ubiquitin ligases in the HBV life cycle

Original language description

Ubiquitination, the covalent attachment of one or more ubiquitin moieties to a target protein catalyzed by E3 ubiquitin ligases, regulates several cellular processes. Depending on its topology and form, ubiquitination influences protein stability, half-life, subcellular localization, and secretion. These mechanisms can be exploited by viruses to facilitate their life cycle. The hepatitis B virus (HBV) is an enveloped, hepatotropic virus that can cause chronic hepatitis B (CHB), acondition affecting more than 254 million people worldwide. HBV depends on host cell autophagy and secretory pathways for effective replication and the production of infectious viral progeny. Nedd4, an E3 ubiquitin ligase, polyubiquitinates the HBV capsid protein (HBc), enabling its recognition by the ESCRT-0 (endosomal sorting complexes required for transport) protein TSG101 and its subsequent sorting to the multivesicular body (MVB), thereby facilitating virion secretion1. Employing mass spectrometry, we searched for novel HBc-interacting host proteins and identified the E2/E3 hybrid enzyme UBE2O and the E3 ubiquitin ligase HUWE12. UBE2O possesses dual activity as an E2 ubiquitin-conjugating enzyme and an E3 ligase. HUWE1, a HECT domain-containing E3 ligase, is implicated in DNA damage repair, the ubiquitin-proteasome system, and autophagy. To investigate the role of UBE2O and HUWE1 in the HBV life cycle, we performed specific siRNA-mediated knockdowns in HBV-infected HepG2-NTCP cells. At 6 d.p, we measured viral parameters, including the levels of HBV RNA/DNA and secreted HBeAg using (RT-)qPCR and ELISA, respectively. Additionally, we analyzed the amount and phosphorylation status of intracellular nucleocapsids, as well as the quantities of secreted viral particles. Compared with HBV-infected cells transfected with a non-specific control siRNA, UBE2O depletion led to a significant decrease in the levels of HBV RNA/DNA and secreted HBeAg. Intracellular capsids and HBc were also significantly reduced upon UBE2O knockdown, suggesting a potential pro-viral role of UBE2O. In contrast, HUWE1 depletion had no significant effect on HBV RNA/DNA levels or secreted HBeAg, but led to amarked accumulation of intracellular HBc/capsids. Notably, the downregulation of either UBE2O or HUWE1 significantly reduced virion secretion, while leaving naked capsid secretion unaffected. Previously, we demonstrated that UBE2O catalyzes multi-monoubiquitination of HBc and viral nucleocapsids3. Mechanistic studies on a potential interaction between HBc/ capsids and HUWE1, as well as HUWE1-mediated ubiquitination of HBc/capsids, are currently ongoing. To further investigate the possible connection between UBE2O-mediated capsid monoubiquitination and the host endosomal secretion pathway, we used confocal microscopy and capsid co-immunoprecipitation. Both UBE2O and HBc colocalized with compartments positive for the MVB marker CD63. Subsequent experiments revealed an increased association between the ESCRT-0 component HGS and capsids upon UBE2O overexpression. Furthermore, an interaction between capsids and the ESCRT-II accessory protein EAP30 occurred exclusively in the presence of UBE2O. Collectively, our data identify UBE2O as a key host factor supporting HBV replication and nucleocapsid formation, and indicate that both UBE2O and HUWE1 promote virion egress.nThe work was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) – Funded by the European Union – NextGenerationEU.

Czech name

—

Czech description

—

Type

O - Miscellaneous

CEP classification

—

OECD FORD branch

10608 - Biochemistry and molecular biology

Project

<a href="/en/project/LX22NPO5103" target="_blank" >LX22NPO5103: National Institute of Virology and Bacteriology</a><br>

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2025

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů