AbstractHBeAg, a non-particulate protein of hepatitis B virus (HBV), is translated from the precore/core region as a precursor, which is post-translationally modified. Subgenotype A1 of HBV, which is a risk factor for hepatocellular carcinoma (HCC), has unique molecular characteristics in the basic core promoter/precore regions. Carriers of A1 exhibit early HBeAg loss. We sought to further characterize the precore proteins of A1 in vitro. HuH-7 cells were transfected with subgenomic constructs expressing individual precore proteins. Western blot analysis using DAKO anti-core antibody showed the expected sizes and a 1 kDa larger band for P22, P20 and P17. Using confocal microscopy, a cytoplasmic accumulation of HBeAg and precursors was observed with P25-expressing plasmid, whereas P22 localized both in the cytoplasm and nucleus. P20 and P17, which lack the carboxy end of P22 showed strong nuclear accumulation, implicating a nuclear localization signal in the N-terminal 10 amino acids. G1862T, unique to subgenotype A1, is frequently found in HBV from HCC patients. P25 with G1862T showed delayed and reduced HBeAg expression/secretion. Knock-out of core in the replication competent clones led to precore protein accumulation in the cytoplasm/perinuclear region, and decreased HBeAg secretion. Knock-out of precore proteins increased HBsAg secretion but intracellular HBsAg expression was unaffected. Over-expression of precore proteins in trans led to decreased HBsAg expression and secretion. Intracellular trafficking of HBV A1 precore proteins was followed. This was unaffected by the CMV promoter and different cell types. In the viral context, precore protein expression was affected by absence of core, and affected HBsAg expression, suggesting an interrelationship between precore proteins, HBcAg and HBsAg. This modulatory role of HBeAg and its precursors may be important in viral persistence and ultimate development of HCC.
Molecular characteristics and stages of chronic hepatitis B virus infection