Mutations in the basal core promoter of HBV lead to intracellular accumulation of viral nucleic acids in vivo

Question:

Mutations in the precore (PC) (G1896A), which abolishes HBeAg production, and in the basal core promoter (BCP) region (A1762T/G1764A) belong to the most common variants in HBeAg negative chronic hepatitis B patients. Moreover, the double mutation BCP/PC has been associated with a more severe clinical course of HBV infection. Aim of this study was to investigate the serological and intracellular characteristics of viruses harboring the BCP mutation either alone or in combination with the PC mutation and in comparison to wildtype (wt) and PC variants in vivo using humanized uPA/SCID/beige (USB) mice.

Methods:

24 mice (6 per group) displaying comparable levels of human liver chimerism were inoculated with cell culture-derived wt HBV (genotype D; 1E7 HBV DNA genome equivalents) or its derived variants (PC, BCP, BCP/PC). Virological parameters were determined by qRT-PCR, immunofluorescence and RNA in situ hybridization. HBsAg and HBeAg were measured by diagnostic kits. Serum pgRNA levels were quantified by qPCR after DNase treatment.

Results:

After achieving stable viral titers, median HBV DNA and pgRNA levels in serum did not differ significantly among groups (HBV DNA/ml = wt:4E8; PC:2E9; BCP:9E8; BCP/PC:5E8; pgRNA/ml = wt:1E8; PC:6E8; BCP:1E8, BCP/PC:1E8). However, intrahepatic analyses revealed that mice infected with BCP and BCP/PC variants harbored higher levels of HBcAg in infected human hepatocytes, higher amounts of pgRNA/hGAPDH (BCP: 4.3 fold and BCP/PC: 2.3 fold) and 10 times higher amounts of rcDNA/cell in comparison to wt and PC viruses (wt:323; PC:330; BCP:3316; BCP/PC:3182), but only slightly higher numbers of cccDNA copies/cell (wt:0.6; PC:0.8; BCP:1.7; BCP/PC:1.1). Consequently, cccDNA activity (rcDNA/cccDNA) appeared 4 and 5 times higher in BCP and BCP/PC groups. The good correlation determined between intrahepatic pgRNA and rcDNA loads suggested that reverse transcription efficiency was similar among groups. The augmentation of intracellular pgRNA was also confirmed by in situ hybridization. Of note, amounts of circulating HBsAg were lower in mice harboring BCP (1879 IU/ml) or BCP/PC (1329 IU/ml) variants in comparison to wt (3532 IU/ml) and PC variants (10160 IU/ml).

Conclusions:

HBV BCP and BCP/PC variants displayed significantly higher intracellular viral activity in vivo in comparison to wt and PC variants. However, the higher intrahepatic viral loads did not translate into increased secretion of virions and HBsAg, indicating occurrence of intracellular HBV accumulation, which may cause additional cell stress and enhanced immune reactions, in part explaining the more severe infection course associated with BCP variants.