Serine Phosphoacceptor Sites within the Core Protein of Hepatitis B Virus Contribute to Genome Replication Pleiotropically

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

Human Hepatitis B Virus Production in Avian Cells Is Characterized by Enhanced RNA Splicing and the Presence of Capsids Containing Shortened Genomes

Experimental studies on hepatitis B virus (HBV) replication are commonly done with human hepatoma cells to reflect the natural species and tissue tropism of the virus. However, HBV can also replicate, upon transfection of virus coding plasmids, in cells of other species. In such cross-species transfection experiments with chicken LMH hepatoma cells, we previously observed the formation of HBV genomes with aberrant electrophoretic mobility, in addition to the those DNA species commonly seen in human HepG2 hepatoma cells. Here, we report that these aberrant DNA forms are mainly due to excessive splicing of HBV pregenomic RNA and the abundant synthesis of spliced DNA products, equivalent to those also made in human cells, yet at much lower level. Mutation of the common splice acceptor site abolished splicing and in turn enhanced production of DNA from full-length pgRNA in transfected LMH cells. The absence of splicing made other DNA molecules visible, that were shortened due to the lack of sequences in the core protein coding region. Furthermore, there was nearly full-length DNA in the cytoplasm of LMH cells that was not protected in viral capsids. Remarkably, we have previously observed similar shortened genomes and non-protected viral DNA in human HepG2 cells, yet exclusively in the nucleus where uncoating and final release of viral genomes occurs. Hence, two effects reflecting capsid disassembly in the nucleus in human HepG2 cells are seen in the cytoplasm of chicken LMH cells

Phosphorylation State-Dependent Interactions of Hepadnavirus Core Protein with Host Factors

Dynamic phosphorylation and dephosphorylation of the hepadnavirus core protein C-terminal domain (CTD) are required for multiple steps of the viral life cycle. It remains unknown how the CTD phosphorylation state may modulate core protein functions but phosphorylation state-dependent viral or host interactions may play a role. In an attempt to identify host factors that may interact differentially with the core protein depending on its CTD phosphorylation state, pulldown assays were performed using the CTD of the duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) core protein, either with wild type (WT) sequences or with alanine or aspartic acid substitutions at the phosphorylation sites. Two host proteins, B23 and I2PP2A, were found to interact preferentially with the alanine-substituted CTD. Furthermore, the WT CTD became competent to interact with the host proteins upon dephosphorylation. Intriguingly, the binding site on the DHBV CTD for both B23 and I2PP2A was mapped to a region upstream of the phosphorylation sites even though B23 or I2PP2A binding to this site was clearly modulated by the phosphorylation state of the downstream and non-overlapping sequences. Together, these results demonstrate a novel mode of phosphorylation-regulated protein-protein interaction and provide new insights into virus-host interactions

Yield Optimisation of Hepatitis B Virus Core Particles in E. coli Expression System for Drug Delivery Applications

An E. coli expression system offers a mean for rapid, high yield and economical production of Hepatitis B Virus core (HBc) particles. However, high-level production of HBc particles in bacteria is demanding and optimisation of HBc particle yield from E. coli is required to improve laboratory-scale productivity for further drug delivery applications. Production steps involve bacterial culture, protein isolation, denaturation, purification and finally protein assembly. In this study, we describe a modified E. coli based method for purifying HBc particles and compare the results with those obtained using a conventional purification method. HBc particle morphology was confirmed by Atomic Force Microscopy (AFM). Protein specificity and secondary structure were confirmed by Western Blot and Circular Dichroism (CD), respectively. The modified method produced ~3-fold higher yield and greater purity of wild type HBc particles than the conventional method. Our results demonstrated that the modified method produce a better yield and purity of HBc particles in an E. coli-expression system, which are fully characterised and suitable to be used for drug delivery applications