Assembly of the enveloped hepatitis B virus (HBV) is initiated by packaging of the RNA pregenome and the viral reverse transcriptase-DNA polymerase into a nucleocapsid. The pregenome is then reverse transcribed into single-stranded minus-polarity DNA, which is subsequently replicated to double-stranded DNA. All replicative intermediates are observable in capsids within infected liver, but only relatively mature nucleocapsids containing partially double stranded DNA are found in secreted virions. This observation suggests that maturation of the genome within the capsid is required for envelopment and secretion. We show that the differential distribution of replicative intermediates between intracellular nucleocapsids and secreted virions is also observable in human hepatoma cells transfected with wild-type HBV genomes. However, nucleocapsids were not enveloped or secreted when they were produced by an HBV genome carrying a missense mutation in the DNA polymerase that eliminates all DNA synthesis. An HBV missense mutant defective in the RNase H activity of the polymerase which allowed minus-strand DNA synthesis but not formation of double-stranded DNA was able to form virion-like particles. These experiments demonstrate that immature nucleocapsids containing pregenomic RNA are incompetent for envelopment and that minus-strand DNA synthesis in the interior lumen of the capsid is coupled to the appearance of a signal on the exterior of the nucleocapsid that is essential for its envelopment.

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...