**4. Epigenetic control of HBV transcription: cccDNA methylation**

Isolation and testing of HBV cccDNA shows methylation not only of HBV DNA integrated in host chromosome [39, 40] but also of HBV cccDNA. Methylation is speculated to affect replicative activity of HBV [38], and this hypothesis was confirmed in the hepatoma cell lines [41] and human liver tissue [15, 16]. HBV cccDNA has three CpG islands which harbor methylation in human liver and hepatoma cell lines [37, 38]. Methylation of HBV in the CpG islands of cccDNA is associated with suppressed transcriptional activity of HBV [15, 16] (for recent reviews, see [17, 18]). Especially, methylation of CpG island II is associated with reduced HBV replicability [42].

Although DNA methyltransferases, i.e., DNMT1, DNMT3a, and DNMT3b, are expressed in normal tissues [45], the level of expression is higher in HCC [46], which may explain the increased levels of methylation in hepatocellular carcinoma (HCC) compared to noncancerous tissues [43, 44]. In addition, since the hepatic expression of DNMT3a and DNMT3b, the de novo methylators, increases with age [47], methylation of HBV may also increase with age [42], which might explain suppressed replicative activity of HBV in the later stage of natural history. Degree of methylation also depends on HBeAg positivity [15, 42] and degree of hepatic fibrosis [42].

The mechanism of HBV DNA methylation is still unknown (**Table 2**). From the specific patterns of methylation in the HBV genome [42], it can be speculated that some kind of molecular chaperon (s) may guide the de novo methylation enzymes to the specific target sequence. HBx may play a role, because it recruits DNMT3A to the regulatory promoters [48]. Small RNAs may be a plausible candidate, as suggested by our in vitro study in which short hairpin RNA induced methylation of the target site in HBV [49].


**Table 2.** Mechanisms of methylation in HBV cccDNA.
