ABSTRACT
In our previous transmission electron microscopic study of hepatitis B virus core antigen in transformed yeast cells, we observed core particles passing through the nuclear pores. We have now analyzed 1,421 nuclear pores in transformed yeast, and conclude that 1) translocation of core particles from the nucleus to the cytoplasm occurs through the nuclear pores; 2) translocation sites are located in the center of nuclear pores; 3) at least 95% of pores are involved in the translocation process; 4) proteins as large as 28 nm in diameter can cross the envelope; 5) translocation does not stop, but rather becomes more active during nuclear division in yeast cells.
Subject(s)
Hepatitis B Core Antigens/ultrastructure , Saccharomyces cerevisiae/virology , Biological Transport, Active , Cytoplasm/ultrastructure , Cytoplasm/virology , Hepatitis B Core Antigens/genetics , Hepatitis B Core Antigens/metabolism , Hepatitis B virus/genetics , Microscopy, Electron , Nuclear Envelope/ultrastructure , Nuclear Envelope/virology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Transformation, GeneticABSTRACT
Transformed yeast cells expressing hepatitis B virus core antigen (HBcAg) were found to accumulate abundant core particles in the same way as human hepatocytes infected with hepatitis B virus (HBV) by the present authors. We, therefore, offer a good model system for studying the dynamics of assembly of HBcAg into core particles. To investigate this problem, we have developed a transformed yeast cell in which expression of HBcAg is highly inducible by deprivation of phosphate in the culture medium. At regular intervals after induction, cells were cryo-fixed and processed for transmission electron microscopy by ultrathin sectioning. After induction, HBcAg activity rapidly increased, becoming several hundred times higher than the initial level after 25 h. The core particles first appeared in the nucleus, then in the cytoplasm, and finally in the vacuole. Core particles passing through nuclear pores from the nucleus to the cytoplasm could be seen. Core particles were either incorporated directly in the vacuole or indirectly by first forming an autophagosome. The core particles were then released into the vacuolar sap, and were digested there. Together with the previous studies, our results suggest that, in human hepatocytes, HBcAg polypeptides are synthesized in the cytoplasm, but are assembled into core particles in the nucleus. The assembled core particles are then transported from the nucleus to the cytoplasm through nuclear pores.
Subject(s)
Hepatitis B Core Antigens/ultrastructure , Saccharomyces cerevisiae/ultrastructure , Cell Line, Transformed , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cryoultramicrotomy , Culture Media , Hepatitis B Core Antigens/metabolism , Humans , Liver/metabolism , Liver/ultrastructure , Microscopy, Electron , Saccharomyces cerevisiae/metabolismABSTRACT
We have constructed a yeast strain that has integrated into its chromosomal ribosomal RNA gene site two copies of Hepatitis B virus surface (HBS) antigen gene under the control of the yeast (Saccharomyces cerevisiae) glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and terminator. The level of expression of HBS gene was low in the strain, but upon chemical and physical mutageneses, in combination with an immunological screening procedure, a mutant clone which expressed HBS protein at a high level was obtained. This mutant strain produces HBS antigen stably under non-selective conditions.
