Hepatitis B Virus (HBV) is a small virus whose genome has only four open reading frames. We argue that the simplicity of the virion correlates with a complexity of functions for viral proteins. We focus on the HBV core protein (Cp), a small (183 residue) protein that self-assembles to form the viral capsid. However, its functions are a little more complicated than that. In an infected cell Cp modulates every step of the viral lifecycle. Cp is bound to nuclear viral DNA and affects its epigenetics. Cp correlates with RNA specificity. Cp assembles specifically on a reverse transcriptase-viral RNA complex or, apparently, nothing at all. Indeed Cp has been one of the model systems for investigation of virus self-assembly. Cp participates in regulation of reverse transcription. Cp signals completion of reverse transcription to support virus secretion. Cp carries both nuclear localization signals and HBV surface antigen (HBsAg) binding sites; both of these functions appear to be regulated by contents of the capsid. Cp can be targeted by antivirals -- while self-assembly is the most accessible of Cp activities, we argue that it makes sense to engage the broader spectrum of Cp function. This article forms part of a symposium in Antiviral Research on “From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story.”

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The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.Hepatitis B virus (HBV) replicates its genome via reverse transcription of a pregenomic RNA (pgRNA). This process ultimately produces a double-stranded DNA with a relaxedcircular conformation (rcDNA) (for a review, see reference 57). A distinguishing feature of HBV reverse transcription is that it occurs within the viral capsid. In the absence of HBV capsids, the HBV polymerase (P) is not able to produce rcDNA genomes from pgRNAs (36,58,61). Observations like these suggest that the capsid participates in reverse transcription.HBV capsids are icosahedra with primarily Tϭ4 symmetry (9,16,19). Each capsid contains 120 dimers of the core protein (16,69), and capsids can assemble in the absence of other viral components (59). A region of the core protein that is a candidate for a role in reverse transcription is the carboxy-terminal region of 34 amino acids, known as the carboxy-terminal domain (CTD). Capsids without the CTD assemble normally but do not support genome replication (8,47,70). The CTD can be attached to the capsid interior at the 5-or quasi-6-fold axes (71). Also, the CTD can bind to nucleic acids in vitro (28). Nearly 50% of the residues in the CTD are arginines, which give the CTD a net positive charge. Fourteen of the sixteen arginines are grouped into four clusters of three or four (see Fig. 2A for the amino acid sequence). This arrangement is conserved among mammalian hepadnaviruses ( Fig. 2A), suggesting it is important for function. Serines at positions 155, 162, and 170 are also conserved and can be phosphorylated in HBV (17,21,40). However, it is not fully understood which features of the CTD (arginine clusters, serine phosphoacceptor sites, or other) contribute to genome replication. The goal of the present study is to understand how each arginine cluster contributes to genome replicat...

The core protein of hepatitis B virus can be phosphorylated at serines 155, 162, and 170. The contribution of these serine residues to DNA synthesis was investigated. Core protein mutants were generated in which each serine was replaced with either alanine or aspartate. Aspartates can mimic constitutively phosphorylated serines while alanines can mimic constitutively dephosphorylated serines. The ability of these mutants to carry out each step of DNA synthesis was determined. Alanine substitutions decreased the efficiency of minus-strand DNA elongation, primer translocation, circularization, and plus-strand DNA elongation. Aspartate substitutions also reduced the efficiency of these steps, but the magnitude of the reduction was less. Our findings suggest that phosphorylated serines are required for multiple steps during DNA synthesis. It has been proposed that generation of mature DNA requires serine dephosphorylation. Our results suggest that completion of rcDNA synthesis requires phosphorylated serines.

Hepatitis B virus synthesizes multiple spliced RNAs that can be reverse-transcribed into viral DNA. We thoroughly characterized the contribution of spliced RNAs to DNA synthesis in transfected cultures of Huh7 and HepG2 cells. We found that up to 50% of DNA within intracellular capsids is derived from five spliced RNAs. Expressing HBV P protein and pgRNA from separate plasmids and the use of the CMV-IE promoter contributes to these high levels of encapsidated DNA derived from spliced RNA. A spliced RNA called Sp1 was the predominant species expressed in both cell lines. All spliced RNAs support the synthesis minus-strand DNA and duplex linear DNA. Only one of the spliced RNAs, Sp14, supported the synthesis of relaxed circular DNA, because splicing removed an important cis-acting sequence (hM) in the other four RNAs. Additionally, we created a variant that was deficient in the synthesis of spliced RNA and supported DNA synthesis at wild-type levels. Our results reinforce and extend the idea that a significant fraction of HBV DNA synthesized under common experimental conditions is derived from spliced RNA. It is important that their presence be considered when analyzing HBV DNA replication in transfected cell cultures.