Both northern blot analysis and real-time polymerase chain beta-catenin inhibitor reaction (PCR) quantification showed that pregenomic/pre-C messenger RNA (mRNA) amounts

of pHBV-mtpreS1, pHBV-mtpreS2, and pHBV-mtS were comparable to those of the control (Fig. 3C). Densitometric quantification of the preS/S mRNA signals revealed a preS1- to preS2/S-mRNA ratio shifted versus much higher preS1 mRNA expression in cells transfected with HBV-mtpreS1 and HBV-mtS genomes compared to WT HBV replicating cells, whereas transfection with HBV-mtpreS2 genome showed amounts of preS/S specific transcripts similar to the control (Fig. 3C). The amounts of HBsAg secreted from cells transfected with each of the three mutated HBV genomes were significantly lower compared with those from WT HBV-replicating cells (Fig. 3D). HBeAg was detected only in the medium of the HBV-mtS transfected cells since the HBV-mtpreS1, HBV-mtpreS2 genomes carried a precore stop codon (Fig. 3D). Real-time PCR quantification of cccDNA molecules in HepG2 cells replicating either the WT this website or any of preS/S mutant HBV showed that the size of the cccDNA pool was significantly increased in HBV mutant transfected cells (Fig. 3D), thus

showing that the unbalanced synthesis of envelope proteins results in an abnormal cccDNA accumulation in the nuclei of the infected hepatocytes. Immunofluorescence experiments were performed to investigate the intracellular localization of S and L proteins synthesized by the three above-mentioned preS/S HBV mutants. In WT-genotype D HBV-transfected cells, S and L proteins showed a diffuse distribution throughout the entire cytoplasm (Fig. 4A). In contrast, in cells transfected with HBV-mtpreS1, HBV-mtpreS2, and HBV-mtS genomes, S and L proteins were both predominantly found in the perinuclear region in a granular distribution (Fig. 4B-D) with a staining pattern typical of proteins retained in the endoplasmic

reticulum (ER). Double-labeling experiments using pDsRed2-ER confirmed the predominant Parvulin localization of each of the three mutant envelope proteins at level of the perinuclear ER (Fig. 4). The main objectives of our study were to evaluate whether important mutations in the preS/S gene had any impact on the amounts of circulating HBsAg and whether this possible effect could be associated with (or be a consequence of) a reduced HBV replicative activity. Our data demonstrate that in patients infected with HBV strains carrying major rearrangements in the preS/S gene, the HBsAg levels are significantly lower compared with patients infected with WT HBV and, interestingly, the lower amounts of HBsAg are not paralleled by reduced levels of serum HBV DNA. Indeed, the viral load was comparable between mutant preS/S and WT HBV–infected patients.