Signals for Bidirectional Nucleocytoplasmic Transport in the Duck Hepatitis B Virus Capsid Protein

Abstract

Hepadnavirus genome replication involves cytoplasmic and nuclear stages, requiring balanced targeting of cytoplasmic nucleocapsids to the nuclear compartment. In this study, we analyze the signals determining capsid compartmentalization in the duck hepatitis B virus (DHBV) animal model, as this system also allows us to study hepadnavirus infection of cultured primary hepatocytes. Using fusions to the green fluorescent protein as a functional assay, we have identified a nuclear localization signal (NLS) that mediates nuclear pore association of the DHBV nucleocapsid and nuclear import of DHBV core protein (DHBc)-derived polypeptides. The DHBc NLS mapped is unique. It bears homology to repetitive NLS elements previously identified near the carboxy terminus of the capsid protein of hepatitis B virus, the human prototype of the hepadnavirus family, but it maps to a more internal position. In further contrast to the hepatitis B virus core protein NLS, the DHBc NLS is not positioned near phosphorylation target sites that are generally assumed to modulate nucleocytoplasmic transport. In functional assays with a knockout mutant, the DHBc NLS was found to be essential for nuclear pore association of the nucleocapsid. The NLS was found to be also essential for virus production from the full-length DHBV genome in transfected cells and from hepatocytes infected with transcomplemented mutant virus. Finally, the DHBc additionally displayed activity indicative of a nuclear export signal, presumably counterbalancing NLS function in the productive state of the infected cell and thereby preventing nucleoplasmic accumulation of nucleocapsids.Viruses that replicate in the nucleus have evolved means to transport their infecting genome through the cytoplasm towards and into the nucleus. As described previously for adenovirus and herpesviruses, viruses typically exploit the preexisting cellular nucleocytoplasmic transport machinery for targeting the nucleocapsid to the nuclear pore, where the genome is released and imported as a more or less complex nucleoprotein, as exemplified by lentiviruses or influenza viruses (12,17,21,42). Nuclear targeting of the nucleocapsid is of particular importance in the case of the hepadnaviruses (hepatitis B viruses [HBVs]), small, enveloped animal viruses, which replicate their circular, partially double-stranded DNA genome only in part in the nucleoplasm. There, the incoming genome matures into an extrachromosomal, covalently closed, circular DNA molecule, the template for genomic RNA synthesis through the cellular transcription machinery (10, 29). The following steps, reverse transcription and plus-strand DNA synthesis, occur inside cytoplasmic nucleocapsids, giving rise to mature core particles. Of these, a fraction is diverted from the major pathway leading to the export of the enveloped virion, thereby serving to establish an elevated intranuclear pool of genome copies early on and to replenish this pool throughout persistent infection (41). For duck HBV (DHBV), it is well established that nuclea...

Central Role of a Serine Phosphorylation Site within Duck Hepatitis B Virus Core Protein for Capsid Trafficking and Genome Release

“…By contrast, the subcellular localization of core was predominantly cytoplasmic for mutants S245A and S245N with individual cells displaying a moderate nuclear core staining. This observation may be due to impaired nuclear import or accelerated nuclear export of core protein (24). Identical results were obtained in LMH cells (data not shown).…”

DNA-tumor virus entry—From plasma membrane to the nucleus

“…These results were similar to studies with duck hepatitis virus (dHBV) (55,202,203). dHBV core proteins have an internal NLS as the carboxy-terminus of the HBV core (204). A dHBV knock-out mutant lacking the core NLS was not found at the NPC suggesting that this NLS was directly or indirectly required for capsid localization to the NPC.…”

Avian hepatitis B viruses: Molecular and cellular biology, phylogenesis, and host tropism

“…This is essential for packaging of the pgRNA into the nucleocapsid as well as progression of reverse transcription within [44,46] . In addition, a nuclear localization signal is present between aa 184 and 226 along with a nuclear export signal [47] . The major phosphorylation sites in the core protein are mapped to the arginine-rich C-terminus.…”