Hepatitis B virus covalently closed circular DNA is associated with methylated histones H3 and H4 and heterochromatin complex proteins : implication of their roles in viral replication

Abstract

Hepatitis B virus covalently closed circular DNA (HBV cccDNA) forms a mini-chromosome structure inside infected hepatocyte nuclei and plays an important role in chronic hepatitis B infection. Methylation of cccDNA-bound histone and the associations of heterochromatin HP1 complex related proteins with cccDNA were investigated in this thesis using transient transfection study system and chromatin immunoprecipitation assay. Di- and tri-methylation of histone H3 lysine 4 residue (H3K4), which plays an activating role in eukaryotic transcription, were found to associate with cccDNA in a way in parallel to the level of HBV replication in our system. On the other hand, tri-methylation of H3K9, which plays an inhibitory role in eukaryotic transcription, was found to associate with cccDNA during decline of HBV replication. During the decline of HBV replication, cccDNA was associated with histone methyltransferases SUV39H1 and SUV420H1 and histone demethylase PLU1. The dynamic of the association of heterochromatin protein 1 (HP1) to cccDNA was similar to that of SUV39H1. The association of cccDNA with five HP1 complex-related proteins (three DNA methyltransferases Dnmt3a, Dnmt3b and Dnmt1 and two methylated DNA binding proteins MBD1 and MeCP2) was studied, and their associations could be roughly divided into two stages. From 72 hours to 96 hours post-transfection, there was an increased association of cccDNA with Dnmt3a, Dnmt3b and MBD1, which was in parallel to the increased association of HP1 and SUV39H1with -cccDNA. From 96 hours to 120 hours after transfection, an increased association of Dnmt1 and MeCP2 with cccDNA was detected, which was correlated to that of SUV420H1. At the time when HBV replication was declining at 120 hours post-transfection, a highest association of SUV39H1, SUV420H1, HP1 and all 5 HP1 complex-related proteins with cccDNA was found. In conclusion, methylation of cccDNA-bound histone was associated with HBV replication. Activating H3K4 methylation was found to correlate with increase in HBV replication, while inhibitory H3K9 methylation correlated with decrease in HBV replication. The association of HP1 was in parallel to that of SUV39H1, indicating that HP1-SUV39H1 complex might be involved, and thereby recruiting other proteins for transcription suppression. Recruitment of DNA methyltransferases and methylated DNA binding proteins to cccDNA provided further evidence that methylation of cccDNA plays a role in transcription suppression. This study identified the associations of methylated histone and other related proteins with cccDNA and their correlations with viral replication. These results enhance our knowledge in HBV replication cycles and transcription regulation. It may show a novel area in development of antiviral drugs such as histone methyltransferase modulators.