Molecular determinants within the C-termini of L-HDAg that regulate hepatitis D virus replication and assembly

Hongbo Guo; Qiudi Li; Chunyang Li; Yao Hou; Yibo Ding; Dan Liu; Yi Ni; Renxian Tang; Kuiyang Zheng; Stephan Urban; Wenshi Wang

doi:10.1016/j.jhepr.2023.100961

Molecular determinants within the C-termini of L-HDAg that regulate hepatitis D virus replication and assembly

JHEP Rep. 2023 Nov 15;6(1):100961. doi: 10.1016/j.jhepr.2023.100961. eCollection 2024 Jan.

Authors

Hongbo Guo¹, Qiudi Li¹, Chunyang Li¹, Yao Hou¹, Yibo Ding¹, Dan Liu¹, Yi Ni^{2

3}, Renxian Tang¹, Kuiyang Zheng¹, Stephan Urban^{2

3}, Wenshi Wang¹

Affiliations

¹ Department of Pathogen Biology and Immunology, Jiangsu Key Laboratory of Immunity and Metabolism, Jiangsu International Laboratory of Immunity and Metabolism, Xuzhou Medical University, Xuzhou, China.
² Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany.
³ German Centre for Infection Research (DZIF), partner site Heidelberg, Heidelberg, Germany.

Abstract

Background & aims: Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus, which confer regulatory roles that are distinct from those of S-HDAg. Notably, these residues are divergent between different genotypes. We aimed to elucidate the molecular determinants within the C-termini that are essential for the regulatory role of L-HDAg in HDV replication and assembly.

Methods: Northern blot, reverse-transcription quantitative PCR, and a newly established HDV trans-complementary system were used in this study.

Results: C-termini of L-HDAg, albeit with high sequence variation among different genotypes, are interchangeable with respect to the trans-inhibitory function of L-HDAg and HDV assembly. The C-terminus of L-HDAg features a conserved prenylation CXXQ motif and is enriched with proline and hydrophobic residues. Abolishment of the CXXQ motif attenuated the inhibitory effect of L-HDAg on HDV replication. In contrast, the enrichment of proline and hydrophobic residues per se does not modify the trans-inhibitory function of L-HDAg. Nevertheless, these residues are essential for HDV assembly. Mechanistically, prolines and hydrophobic residues contribute to HDV assembly via a mode of action independent of the prenylated CXXQ motif.

Conclusions: Within the C-terminus of L-HDAg, the CXXQ motif and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly. This intrinsic viral regulatory mechanism we elucidated deepens our understanding of the unique life cycle of HDV.

Impact and implications: Hepatitis D virus (HDV) encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus. This C-terminal extension in L-HDAg confers regulatory roles in the HDV life cycle that are distinct from those of S-HDAg. Herein, we found that C-termini of L-HDAg, although with high sequence variation, are interchangeable among different HDV genotypes. Within the C-terminus of L-HDAg, the prenylation motif, and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly.

Keywords: Hepatitis B virus; Hepatitis D virus; L-HDAg; Virion assembly and production; Virus replication.