[Subcellular distribution and translocation of hepatitis B virus core protein in HepG2.2.15 cells].
OBJECTIVE
The hepatitis B virus core protein has been found in nuclei, cytoplasm, or both of hepatocytes transfected with HBV DNA. It is still unclear whether intact core particles could pass through nuclear pores and what could be the mechanism regulating the subcellular localization of the core protein. This study on the distribution of core protein in hepatocytes and its translocation has a potential advantage to learn more about the HBV life cycle.


METHODS
Dimethyl sulphoxide (DMSO, 2%), which effects hepatic differentiation, and/or 1 micro mol/L heteroaryldihydropyrimidine Bay41-4109, which interferes with the assembly of core particles, were added into HepG2.2.15 cell culture system for 4 days. The hepatitis B virus core antigen (HBcAg) and hepatitis B virus surface antigen (HBsAg) were stained with fluorescent immunocytochemistry and then observed under a confocal microscope. HBcAg in cytoplasm and nuclei were respectively extracted and analyzed using Western blot. HBV covalently closed circular DNA (cccDNA) was detected by using selective PCR method.


RESULTS
The HBcAg was mostly expressed in the cytoplasm and weak signals of cccDNA were detected in the control HepG2.2.15 cells. After DMSO treatment, the expression of HBcAg in cytoplasm was increased about 2.5-fold; the expression of HBcAg and cccDNA in nuclei also increased. With the use of Bay41-4109, the signal of HBcAg in cytoplasm decreased 2/3, but it increased in the nuclei, and cccDNA decreased in the nuclei. When the HepG2.2.15 cells were treated both with DMSO and Bay41-4109, cord-liked distribution of HBsAg was observed in the cytoplasm. HBcAg in cytoplasm was decreased 1/2 but the HBcAg in the nuclei increased about 5-fold, whereas the cccDNA was almost negative.


CONCLUSIONS
In HepG2.2.15 cells, the core protein is mainly assembled as a formation of core particles in the cytoplasm and they are blocked by the nuclear membrane. Bay41-4109 interferes with the assembly of core particles and the dissociated core proteins are able to enter the nuclei. DMSO promotes the nuclear entry of core protein/core particles and facilitates the formation of cccDNA.
Pan
Xiao-ben
XB
Peking University People's Hospital, Peking University Hepatology Institute, Beijing 100044, China.
Han
Jin-chao
JC
Wei
Lai
L
Peng
Dan-dan
DD
Gao
Yan
Y
D045584:Chromosome Positioning; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D056945:Hep G2 Cells; D006512:Hepatitis B Core Antigens / Q000378:metabolism*; D006515:Hepatitis B virus / Q000502:physiology*; D006801:Humans; D009362:Neoplasm Metastasis; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D014758:Viral Core Proteins / Q000378:metabolism*; D019065:Virus Assembly
C464059:BAY 41-4109; D006512:Hepatitis B Core Antigens; D011725:Pyridines; D011743:Pyrimidines; D014758:Viral Core Proteins; D004121:Dimethyl Sulfoxide