Hepatitis B virus (HBV) is a major human pathogen causing acute and chronic liver inflammation. It is the prototype of a family of hepatotrophic, enveloped DNA viruses with a very narrow host range, referred to as hepadnaviridae. The spherical virus particle has a diameter of 42 nm and consists of an envelope carrying three surface proteins which surrounds an icosahedral capsid enclosing an open circular, partially double stranded, 3.2-kb DNA as well as the viral DNA polymerase. The capsid has a diameter of 30 nm and is formed by multiple copies of one species of core protein (for a review, see reference 22). This protein comprises 185 amino acids (aa) for genotype A and forms dimers which self-assemble in heterologous expression systems into shells of Tϭ3 and Tϭ4 symmetry (32). The C-terminal 30 aa are very rich in arginine residues and probably bind to the encapsidated viral nucleic acid. The N-terminal 155 aa are sufficient for capsid formation (9) and referred to as the assembly domain. The fold of the assembly domain in core particles has been determined by electron cryomicroscopy (4, 8). The capsid is a very immunogenic antigen (HBV core antigen [HBcAg]), and the corresponding antibody (anti-HBc) mainly binds to a conformation-dependent epitope. An antibody with a different specificity (anti-HBV e antigen [anti-HBe]) which binds also to denatured core protein (24) is formed by only a fraction of infected individuals.During HBV infection the nucleocapsid is released from the incoming virus into the cytosol, and the viral DNA genome is transported into the nucleus and repaired to give a circular covalently closed episome. This DNA serves as the template for transcription by host factors. A 3.5-kb RNA has two functions: (i) it is the mRNA for translation of core protein and reverse transcriptase/DNA polymerase (P protein), and (ii) it is bound by P protein and packaged by multiple copies of core protein dimers into capsids. The viral DNA genome is then synthesized in the lumen of the capsid by reverse transcription of the 3.5-kb RNA followed by second-strand DNA synthesis. The nucleocapsid can follow two different pathways. It can stay within the cell, in which case its genome contributes to the intracellular amplification of the viral episomes (27). Alternatively, nucleocapsids interact at intracellular membranes of a pre-Golgi compartment with cytosolic domains of viral envelope proteins (5, 23) which are expressed as transmembrane peptides from 2.1-and 2.4-kb mRNAs. This interaction probably initiates and drives budding, resulting in the formation of virions in the lumen of the exocytotic compartment which are released from the cell by secretion. The destiny of nucleocapsids, either disintegration and release of the genome or envelopment, is regulated. In the duck hepatitis B virus animal model, it was demonstrated that early in infection disintegration and genome amplification prevail, whereas later genome amplification ceases (19) and envelopment of capsids leads to formation of virions. Another step dur...
We generated a large number of mutations in the hepatitis B virus (HBV) core gene inserted into a bacterial expression vector. The new mutagenesis procedure generated deletions and insertions (as sequence repeats) of various lengths at random positions between M1 and E145 but not substitutions. The R-rich 30-amino-acid C-terminal domain was not analyzed. A total of 50,000 colonies were tested with a polyclonal human serum for the expression of hepatitis B core or e antigen. A total of 110 mutants randomly chosen from 1,500 positive colonies were genotyped. Deletions and insertions were clustered in four regions: D2 to E14, corresponding to the N-terminal loop in a model for the core protein fold (B. Bottcher, S. A. Wynne, and R. A. Crowther, Nature 386:88-91, 1997); V27 to P50 (second loop); L60 to V86 (upper half of the alpha helix forming the N-terminal part of the spike and the tip of the spike); and V124 to L140 (C-terminal part of the C-terminal helix and downstream loop). Deletions or insertions in the remaining parts of the molecule forming the compact center of the fold seemed to destabilize the protein. Of the 110 mutations, 38 allowed capsid formation in Escherichia coli. They mapped exclusively to nonhelical regions of the proposed fold. The mutations form a basis for subsequent analysis of further functions of the HBV core protein in the viral life cycle.
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