STRUCTURE AND ASSEMBLY OF THE HEPATITIS B NUCLEOCAPSID PROTEIN

背景：乙型肝炎病毒（HBV）感染是一种疾病

Background Hepatitis B Virus (HBV) infection is a worldwide biomedical problem and an improved understanding of the assembly and structure of the virus may help develop new antiviral therapies. The HBV core gene codes for two products: (a) the 183 residue core antigen (HBcAg) which forms nucleocapsid particles which encapsidate the viral DNA and a multifunctional HBV polymerase; (b) a precore protein (pre-C) which is N- and C- terminally processed to form a secreted non-particulate protein called e- antigen (HBeAg). HBcAg has been expressed in E.coli were it assembles in the bacterial cytoplasm into icosahedral capsids, which contain bound host nucleic acid. Deletion of the polybasic C-terminal 34 residues also produces assembly competent protein. The C-terminal truncated protein (Cpe: residues 1-149) does not contain nucleic acid, can be highly purified and is more suitable for structural studies than the full-length protein. Native HBeAg is also C-terminally truncated at position 149 and in addition contains a 10 residue N-terminal extension derived from partial processing of pre-C. This protein and several variants have also been expressed in E.coli for structural studies. Results In earlier work, the structure of Cpe was determined by cryo- electron microscopy and image analysis to a resolution of about 0.8 nm. This allowed various structural domains to be discerned, especially a four alpha-helical bundle which formed the surface projections of the capsid. These surface projections were shown by labeling with monoclonal antibodies to be the site of the immunodominant epitope, an important serological marker. To localize other regions of functional and structural importance, we have introduced by site-directed mutagenesis reactive cysteine residues at various sites in the protein that can be specially labeled with electron dense reagents. We previously localized the C-terminal region, an important determinant of capsid assembly and morphogenesis, and using similar approaches are continuing to map other functionally important regions, including the N-terminal domain which also appears to play a role in assembly. After several years of continuous effort, conditions for the crystallization of Cpe have been worked out. Although crystallization of Cpe had been achieved last year, the crystals were difficult to reproduce, were small, and took several months to form. Furthermore, they could not be frozen, a prerequisite for high-resolution studies using a synchrotron light source. Capsids prepared using a different purification strategy, can now be routinely crystallized in less than a week to from large crystals (<0.5 mm) and which can be transferred cyropreservants for successful freezing. Significance and future direction: The high-resolution structure determination of the HBV capsid using X-ray diffraction appears possible using crystals prepared using the methods developed. The 0.9 nm model derived from cryo- electron microcopy will play a major role in the structural work by providing a suitable molecular phasing model. Other targets of HBV for structural determinations include the HBV polymerase and HBeAg. These proteins, or functional regions thereof, will be expressed in E.coli and structure determinations carried out. Summary The Hepatitis B Virus (HBV) is the major worldwide cause of cancer. Although a vaccine is available, chronic HBV is often acquired in childhood. The HBV nucleocapsid plays an important structural role and metabolic role in the life cycle of the virus. An understanding of the molecular structure of the HBV nucleocapsid would allow targeted drug discovery with the aim of preventing the assembly and formation of the virus.