Hepatitis B Virus Genotype C Isolates with Wild-Type Core Promoter Sequence Replicate Less Efficiently than Genotype B Isolates but Possess Higher Virion Secretion Capacity

Abstract

Infection by hepatitis B virus (HBV) genotype C is associated with a prolonged viremic phase, delayed hepatitis B e antigen (HBeAg) seroconversion, and an increased incidence of liver cirrhosis and hepatocellular carcinoma compared with genotype B infection. Genotype C is also associated with the more frequent emergence of core promoter mutations, which increase genome replication and are independently associated with poor clinical outcomes. We amplified full-length HBV genomes from serum samples from Chinese and U. S. patients with chronic HBV infection and transfected circularized genome pools or dimeric constructs of individual clones into Huh7 cells. The two genotypes could be differentiated by Western blot analysis due to the reactivities of M and L proteins toward a monoclonal pre-S2 antibody and slightly different S-protein mobilities. Great variability in replication capacity was observed for both genotypes. The A1762T/G1764A core promoter mutations were prevalent in genotype C isolates and correlated with increased replication capacity, while the A1752G/T mutation frequently found in genotype B isolates correlated with a low replication capacity. Importantly, most genotype C isolates with wild-type core promoter sequence replicated less efficiently than the corresponding genotype B isolates due to less efficient transcription of the 3.5-kb RNA. However, genotype C isolates often displayed more efficient virion secretion. We propose that the low intracellular levels of viral DNA and core protein of wild-type genotype C delay immune clearance and trigger the subsequent emergence of A1762T/G1764A core promoter mutations to upregulate replication; efficient virion secretion compensates for the low replication capacity to ensure the establishment of persistent infection by genotype C.The hepatitis B virus (HBV) is a hepatotropic enveloped DNA virus. The virion-associated relaxed circular DNA is converted to covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes to serve as a template for viral gene expression and genome replication. The four genes are arranged on this 3.2-kb circular genome in the order of core, polymerase (P), surface, and X, with the P gene overlapping with the other three. Besides the core, P, small envelope (S), and hepatitis B X (HBx) proteins, the extra in-frame AUG codons upstream of the surface and core genes permit the expression of large (L) and middle (M) envelope proteins as well as the precore/core protein. The precore/core protein is processed by proteolytic cleavage and secreted as hepatitis B e antigen (HBeAg). The seven viral proteins are translated from five mRNAs generated from the cccDNA template: the 3.5-kb precore RNA for HBeAg, the slightly shorter 3.5-kb pregenomic (pg) RNA for core and P, and subgenomic RNAs of 2.4 kb for L protein, 2

In Vitro Studies Show that Sequence Variability Contributes to Marked Variation in Hepatitis B Virus Replication, Protein Expression, and Function Observed across Genotypes

“…One limitation of our study is that we only investigated one clone per genotype, although we note that our findings pertaining to replication of genotypes B2 and C2 were similar to those of a previous study (10). In contrast, another earlier study observed greater replication in genotype B2 than in genotype C2 (12). Taken together, these and our present studies suggest that it is not possible to make broad generalizations about HBV replication phenotype across genotypes and subtypes.…”

“…A more recent study utilizing 1.28-fold copies of the HBV genome concluded that the HBV genotype D replicated at lower levels than the HBV genotype A, although HBV replication was again low in this study (11). Direct comparisons of the replication phenotype of genotypes B and C utilizing 1.24-mer clones (10) or tandem dimers (12) identified differences in the comparative replication phenotypes of genotypes B and C in these studies, with Sugiyama et al (10) identifying higher levels of DNA replication for genotype C than for genotype B, in contrast to the findings of Qin et al (12), who identified higher levels of DNA replication for genotype B. Genotype differences in virus secretion were also observed, with Qin et al observing higher levels of secretion for genotype C than for genotype B (12), in contrast to Sugiyama et al, who observed similar levels of virion secretion for genotypes B2 (Ba) and C (10). This suggests that it is difficult to generalize about the impact of replication phenotype observed in vitro on the differences in HBV natural history observed across HBV genotypes and subtypes.…”

Unusual Features of Sodium Taurocholate Cotransporting Polypeptide as a Hepatitis B Virus Receptor

“…Particles in the pellet were resuspended in 1/100 the original volume with complete medium or phosphate-buffered saline (PBS) for storage at Ϫ80°C, and the genome copy number was determined by dot hybridization using serial dilutions of cloned HBV DNA of known concentration for calibration (30). Similar procedures were used to purify sHBV from viremic serum samples collected from chronic HBV carriers from Shanghai, China, for another study (31). Informed consent was obtained from patients, and the study was approved by Institutional Board and by the Recombinant DNA Committee of Rhode Island Hospital.…”

“…Covalently closed circular DNA (cccDNA) was extracted according to an established method (33). Virions were immunoprecipitated from culture supernatant by a combination of rabbit antipreS1 antibody (R271) and horse anti-Ad/Ay antibody according to established protocols (29,31). Virion DNA, intracellular replicative DNA, and cccDNA were detected by Southern blotting, while HBV RNA was detected by Northern blotting.…”

“…The HBsAg/ HBeAg ratio refers to HBsAg titer in culture supernatant divided by HBeAg titer from the same volume of culture supernatant. Southern and Northern blot analyses were performed as previously described (29,31), and for such purposes the cells were seeded in 6-or 12-well plates for infection experiments. To avoid bias in detecting ccHBV or sHBV, the 3.1-kb HBV DNA of genotypes B, C, and D was mixed at 1:1:2 ratio for probe labeling.…”