Characterization Of HBV Covalently Closed Circular DNA (cccDNA) Methylation And Its Regulation On Viral Replication And Gene Expression

Despite the widespread use of the HBV vaccine, Hepatitis B virus (HBV) infection remains a major public health problem. HBV belongs to hepatotropic DNA virus. Once in the nuclei of the infected cell, the covelent closed circular DNA is formed, serving as the template for pg RNA production, which is crucial to HBV life cycle. HBV infection triggers the upregulation of DNA transmethylase in the host cell, which causes DNA methylation that regulate the transcription activity of HBV cccDNA. To better understand the mechanism of DNA methylation mediated repression of viral transcription and replication, the following studies were performed.In the first section, we analyzed the distribution of CpG islands within HBV genomes among A-J genotypes. By using the software MethPrimer, three CpG islands were identified in HBV genome, indicating that the HBV DNA can be methylated. CpG island Ⅰ spans the start site of S gene. CpG island Ⅱ overlaps the enhancer Ⅰand the X gene promoter, also in close and CpG island Ⅲ encompasses the start site of the P gene and promoter sp1.Our study suggested that heterogeneity exists in the CpG sites distribution within HBV sequences of different genotypes. Genotypes C, F, G, and H HBV sequences tend to contain two CpG islands, while most strains of genotypes A, B, D, E, and I contain three CpG islands, as previously reported. While three novel CpG islands were identified in genotype B、C、D and H genotype HBV genomes. The CpG dinucleotide density is relatively low in genotype C, and the absence of CpG island I is more frequent in genotype C, when compared with genotype B HBV sequences.In the second section, we obtained the distribution of CpG methylation within HBV cccDNA in the liver biopsy tissues from 30 chinese chronic hepatitis B patients. Patients included in the study had no history of antiviral therapy for HBV. HBV cccDNA were extracted from the liver tissues, and underwent the bisulfite convertion, from which the three CpG islands were PCR amplified, respectively. Our profile revealedthat HBV cccDNA were methylated in the liver of the CHB patients. In our profile, CpG island Ⅰ were barely methylated, while a very low level of methylation can be observed in 5 patients (18.5%) of the study. Also island Ⅱ methylation is associated with suppressed serum HBV DNA titers, The degree of CpG island Ⅱ methylation was significantly higher in patients with lower HBV DNA titer (Log HBV DNA<5) than in that with high HBV DNA titer (Log HBV DNA>5), indicating that the methylation in CpG island Ⅱ represses viral replication, which might be associated with the altered core promoter activity. Age is also an correlation factor, since the methylation rate of the CpG island Ⅱ were significantly higher in patients with age≥40. And our profile also showed that the methylation rate of the CpG island Ⅱ in genotype C patients were significantly higher than that of genotype B patients, while the methylation rate of the CpG island Ⅱ in HBeAg negative patients were significantly higher than that of HBeAg positive patients. The methylation rate of the CpG island Ⅱ in fibrosis stage S3-4 patients were significantly higher than that of SO-2 patients.Furthermore, we found a negative correlation between HBsAg level and the methylation rate of the CpG island Ⅲ detected in the HBV cccDNA samples of the same patients.In the third section, we studied the function of single HBV CpG island in vitro. So far, in vitro HBV DNA methylation study were carried out by using DNA methyltransferase treating the 3.2kb HBV linear monomer, followed by transfection into the HepG2 cells, and detecting the transcription level of viral mRNA by real-time PCR. The study revealed that DNA methylation represses the viral mRNA transcription. However, the function of each CpG island in the DNA methylation mediated regulation of transcription remains unclear. In the present study, the CpG islands were PCR amplified, methylated in vitro, ligated to the complementary DNA fragments, respectively. The circularized monomeric HBV genome containing non-methylated or methylated CpG islands were transfected into HepG2 cells. The results of Northern blot and Southern blot demonstrated that the transcription and replication level of the HBV monomer containing the methylated CpG island II were significantly decreased, when compared to the control group, indicating the CpG island II methylation downregulate the transcriptional activity of HBV cccDNA. Moreover, HBV core protein has also been implicated in the alteration of the chromatin structure of viral minichromosome, binding to CpG islands to regulate the transcriptional activity of cccDNA. We transfected the HepG2 cells with the linear HBV monomer with or without core expression, and the results showed the HBV RNA transcription level did not decrease in the absence the core peotein. Therefore, our profile suggested the HBV core protein may not affect the transcription activity of cccDNA.