Hepatitis B virus (HBV) consists of a glycoprotein envelope and an inner nucleocapsid core which is composed of a single protein, the core antigen (HBcAg). The nucleocapsid encloses a reverse transcriptase and a partially double-stranded DNA genome. 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 nucleocapsid protein (about 21 kDa) is well expressed in E.coli where 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 protein which forms capsid structures when expressed in E. coli (Cpe). The C-terminal truncated protein does not bind host nucleic and can be purified to a high degree of purity using a novel protein purification method which we have developed. Detailed structural analysis of the capsids by physiochemical methods have enabled a 1.7 nm structure to be determined by cryo-electron microscopy and image analysis. One of the main goals of this project remains the preparation of high quality crystals suitable for X-ray diffraction analysis. We have prepared many different mutant forms of Cpe designed to aid in crystal production. Although crystals have been produced which diffract to 0.5 nm, we are trying to produce better crystals which will enable a high resolution structure (<0.3 nm) to be determined. Recently, mutants containing N-terminal extensions have been prepared which although appear assembly incompetent have the correct native conformation. These mutants may provide a simplified route to obtaining X-ray quality crystals and may allow structural analysis by high resolution NMR methods.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Intramural Research (Z01)
Project #
1Z01AR041117-01
Application #
2452789
Study Section
Special Emphasis Panel (PEL)
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
1996
Total Cost
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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