Lysine-71 in the large delta antigen of hepatitis delta virus clade 3 modulates its localization and secretion

Hepatitis delta virus (HDV) is an RNA virus and eight clades of HDV have been identified. HDV clade 3 (HDV-3) is isolated only in the northern area of South America. The outcome of HDV-3 infection is associated with severe fulminant hepatitis. Variations in the large delta antigen (LDAg) between HDV clade 1 (HDV-1) and HDV-3 have been proposed to contribute to differences in viral secretion efficiency, but which changes might be relevant remains unclear. The control of subcellular localization of LDAg has been reported to be associated with post-translational modifications, such as phosphorylation and isoprenylation. We have observed evidence for acetylation on the LDAg of HDV-3 (LDAg-3) and LDAg of HDV-1 (LDAg-1). Green fluorescent protein-fused LDAg-3 (GFP-LD3) was used to investigate the cellular distribution and secretion of the protein. Sequence alignment of LDAg amino acids suggested that lysine-71 of LDAg-3 could be an acetylation site. Expression of a mutant form of LDAg-3 with an arginine-substitution at lysine-71 (GFP-LD3K71R) showed a distribution of the protein predominantly in the cytoplasm instead of the nucleus. Western blot analyses of secreted empty viral particles (EVPs) revealed a higher amount of secreted GFP-LD3K71R compared to GFP-LD3. Furthermore, the ectopic expression of p300, a histone acetyltransferase, led to a reduction of GFP-LD3 in EVPs. By contrast, expression of three histone deacetylases (HDAC-4, -5, and -6) facilitated the secretion of GFP-LD3. Combined, our observations support the hypothesis that the acetylation status of LDAg-3 plays a role in regulating LDAg-3's localization inside the nucleus or cytoplasm, and its secretion.