Role of Hepatitis B core and X proteins in HBV transcription and recruitment of histone modifying enzymes to HBV covalently closed circular DNA

Abstract

The hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) exists as a minichromosomal conformation and is the transcriptional template of all HBV RNA. During HBV replication, both the hepatitis B core (HBc) and X (HBx) proteins are recruited to the cccDNA minichromosome. Both proteins play multiple roles in viral replication. HBc has been demonstrated to be a component of the cccDNA minichromosome and exert epigenetic regulation onviral transcription. HBx is also suggested to associate with cccDNA and play a role in the maintenance of HBV replication. This thesis aimed to identify specific amino acid residue(s) of HBc and HBx that are responsible for their interaction with cccDNA and their roles in transcriptional regulation. For the study of HBc, 17 HBc mutants with either substitution of arginine to alanine residues or replacement of nearby residue to bulky phenylalanine in the C-terminal region (amino acid 150-181) were created. While for HBx, 30 mutants with consecutive amino acid substitutions at the HBx transactivating domain (amino acid 51-154) expression plasmids were created. These HBc or HBx expression plasmids, in wild-type or mutant forms, were transiently co-transfected with HBc-null or HBx-null plasmid-free HBV DNA into hepatoma cells. The effects of HBc and HBx mutations on viral replication were assessed by measuring the levels of nuclear circularized HBV DNA, intracellular encapsidated HBV DNA, total HBV RNA, and secretory HBsAg. Furthermore, the recruitment levels of the histone modifying enzymes towards cccDNA minichromosome were assessed by measurement of association degree between specific enzymes and cccDNA. In this plasmid-free HBV DNA transient transfection system, a complete HBV replication cycle, starting from cccDNA-initiated transcription to the secretion of HBV viral antigens, could be demonstrated. Compared with the wild-type HBc strain, HBc mutants with mutations in arginine cluster III and IV (amino acid 164-167; 172-175 respectively) had significantly lower levels of total HBV RNA, encapsidated HBV DNA, and secretory HBsAg. Similarly, a lower-than-wild-type level of total HBV RNA, encapsidated HBV DNA, secretory HBsAg was also detected in the HBx mutants with mutations at amino acid 55-64 and 121-126.Chromatin immunoprecipitation experiments revealed that both these HBc and HBx mutants had a lower degree of association with cccDNA when compared with wild-type. Furthermore, the recruitment of histone acetyltransferases (HATs) to cccDNA in the HBc arginine clusters III and IV mutants was lower than that of wild-type. For the HBx mutants, in addition to the reduced recruitment of HATs, an augmented recruitment of histone deacetylases (HDACs) was observed in HBx mutants with mutations at amino acid 55-64 and 121-126. In conclusion, this study presented the importance of specific HBc amino acid residues (164-167 and 172-175) and HBx amino acid residues (55-60and 121-126) in regulating viral transcription via their interaction with cccDNA and modulation of the recruitment of histone modifying enzymesto cccDNA. The identification of key domains of HBc and HBx may provide insights to the identification of novel therapeutic targets against HBV infection.