Aris Haryanto, AndrÃÆÃÂ© Schmitz, Birgit Rabe, Evelyn Gassert Angelika Vlachou, and Michael Kann
Capsid protein of hepatitis B virus (HBV) consists of a single karyophilic protein species. It mediates the entry of the viral DNA into the nucleus using the cellular transport receptors importin �?± and �?�?. The nuclear localization signal (NLS) is localized within the C-terminus of the HBV capsid protein, overlapping with eight phosphorylation sites. We first investigated the NLS in more detail observing that the amino acid sequence PRRRTPSPRRR is sufficient to mediate efficient nuclear import of bovine serum albumin. Phosphorylation of this sequence blocked its transport capacity. However, empty E. coli-expressed capsids that exposed the C-terminus on their surface failed to interact with the nuclear envelope in digitonin-permeabilized cells unless becoming phosphorylated. Consistently, treatment of cells with a protein kinase inhibitor prevented nuclear localization of a fusion protein of EGFP and capsid protein. The results show that phosphorylation is required for nuclear transport of the HBV capsid protein but that phosphorylation must occur outside the transport signal, a phosphorylation pattern that is similar to that observed for the SV40TAg. Moreover, it implies a complex regulation of phosphorylation and dephosphorylation during the hepadnaviral life cycle.
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