The nuclear localization signal of Hepatitis B Virus Core Protein : characterization by expression as EGFP-Core Fusion Protein
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HBV core particles are involved in a number of important functions in the replication cycle of HBV. Nuclear import studies of cellular protein showed that HBV capsid binds to the nuclear pore complex, following the classical pathway of karyophilic proteins, which is mediated by the cellular proteins importin alpha and beta. The transport of the capsid into the nucleus is facilitated by a nuclear localization signal that is located within the carboxyl terminal domain between amino acid 158 and 168 of the core protein. Unlike the NLS consensus sequence, HBV core NLS does not comprise a lysine residue, leading to the assumption that the NLS shows only weak interaction with importin alpha. The understanding of the NLS and its nuclear transport receptor interaction is mainly based on observations using biochemical assays like co-immune precipitation and in vitro transport assays. This study was intended to provide corresponding in vivo evidence. By expressing fusion proteins of core and fluorescent marker protein, the strength of core NLS and the impact of phosphorylation was investigated. To analyze the intracellular localization of HBV capsid in human liver cells, firstly the entire HBV genome was transfected into HuH-7 cells. The HBV capsid was found to be localized predominantly in the nucleus as it is in vivo. EGFP was chosen as a fluorescent marker protein to generate fusion protein with HBV core. The fusion protein was shown to form at least dimers but failed to assemble to capsids. This fusion protein was imported into the nucleus, redundancy of the HBV core NLS increased the translocation. Moreover the replacement of the theoretically weak HBV core NLS by the NLS of the SV40 Tag led to a similar transport competence.The nuclear import was dependent upon phosphorylation as shown by Staurosporine treatment of transfected cells. The different fusion proteins used in these assays showed that serine 141 is the key amino acid that has to be phosphorylated. This phosphorylation however seems to differ in different cell lines and was apparently affected by the growth conditions of the cells. It must be thus concluded that there is a complex phosphorylation pattern of the core protein that affects different steps in the viral life cycle including nuclear transport. The coordination of these different phosphorylation steps remains to be elucidated.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Wettenberg : VVB Laufersweiler 2006