Chronic hepatitis B virus (HBV) infection is a global public health challenge on the same scale as tuberculosis, HIV, and malaria. The International Coalition to Eliminate HBV (ICE-HBV) is a coalition of experts dedicated to accelerating the discovery of a cure for chronic hepatitis B. Following extensive consultation with more than 50 scientists from across the globe, as well as key stakeholders including people affected by HBV, we have identified gaps in our current knowledge and new strategies and tools that are required to achieve HBV cure. We believe that research must focus on the discovery of interventional strategies that will permanently reduce the number of productively infected cells or permanently silence the covalently closed circular DNA in those cells, and that will stimulate HBV-specific host immune responses which mimic spontaneous resolution of HBV infection. There is also a pressing need for the establishment of repositories of standardised HBV reagents and protocols that can be accessed by all HBV researchers throughout the world. The HBV cure research agenda outlined in this position paper will contribute markedly to the goal of eliminating HBV infection worldwide.

Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.

The heat shock protein Hsp9O is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp9O interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp9O pathway in the formation of an RNP.Hepatitis B viruses (hepadnaviruses) are a group of small DNA viruses that replicate through a reverse transcription pathway. Reverse transcription in these viruses is initiated by a protein-priming mechanism as depicted in Fig. 1. The virally encoded reverse transcriptase binds to a short RNA sequence located at the 5' end of the viral pregenomic RNA (the template for reverse transcription), termed £, and initiates DNA synthesis de novo by using a tyrosine residue within the polymerase polypeptide as the primer (the protein-priming reaction) (2, 3). In addition, binding of the reverse transcriptase to e triggers nucleocapsid assembly, thus ensuring packaging of the RNA pregenome and the polymerase into nucleocapsids where reverse transcription takes place (4, 5). Therefore, the formation of a specific ribonucleoprotein (RNP) complex between the reverse transcriptase and s is critical for viral assembly and DNA synthesis (1, 4, 6-10).Since the RNP complex between the reverse transcriptase and £ RNA is transient (1, 7), its formation most likely depends on a particular conformation of the polymerase that is competent for binding to s. An important question is how the polymerase is stabilized in such a conformation that allows for formation of the RNP complex. To address this problem, we have exploited a recently developed cell-free system that permits expression of an enzymatically active reverse transcriptase of an avian hepadnavirus, the duck hepatitis B virus (DHBV), in a rabbit reticulocyte lysate (RL) (2, 3, 7). With the help of this system, we have identified a molecular chaperone, the heat shock protein Hsp9O, as an essential host factor required for the polymerase activity in vitro and in vivo. Hsp90 binds to the polymerase and facilitates the interaction between the polymerase and the £ RNA and thus plays an essential role in initiation of viral DNA synthesis and RNA packaging. MATERIALS AND METHODSPlasmids. Plasmids pHP and pHTP for expression of the DHBV polymerase in vitro have been described (3). pT3.1118 (11), used for in vitro expression of the glucocorticoid receptor (GR), was provided by K. Yamamoto (University of California, San Francisco). pCMV-DHBV directs expression of the DHBV pregenome from the cytomegalovirus immediate early promoter (12, 13). pTet-DHBV was derived from pCMVThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

As a para-retrovirus, hepatitis B virus (HBV) is an enveloped virus with a double-stranded (DS) DNA genome that is replicated by reverse transcription of an RNA intermediate, the pregenomic RNA or pgRNA. HBV assembly begins with the formation of an “immature” nucleocapsid (NC) incorporating pgRNA, which is converted via reverse transcription within the maturing NC to the DS DNA genome. Only the mature, DS DNA-containing NCs are enveloped and secreted as virions whereas immature NCs containing RNA or single-stranded (SS) DNA are not enveloped. The current model for selective virion morphogenesis postulates that accumulation of DS DNA within the NC induces a “maturation signal” that, in turn, triggers its envelopment and secretion. However, we have found, by careful quantification of viral DNA and NCs in HBV virions secreted in vitro and in vivo, that the vast majority of HBV virions (over 90%) contained no DNA at all, indicating that NCs with no genome were enveloped and secreted as empty virions (i.e., enveloped NCs with no DNA). Furthermore, viral mutants bearing mutations precluding any DNA synthesis secreted exclusively empty virions. Thus, viral DNA synthesis is not required for HBV virion morphogenesis. On the other hand, NCs containing RNA or SS DNA were excluded from virion formation. The secretion of DS DNA-containing as well as empty virions on one hand, and the lack of secretion of virions containing single-stranded (SS) DNA or RNA on the other, prompted us to propose an alternative, “Single Strand Blocking” model to explain selective HBV morphogenesis whereby SS nucleic acid within the NC negatively regulates NC envelopment, which is relieved upon second strand DNA synthesis.