Replication advantage and host factor-independent phenotypes attributable to a common naturally occurring capsid mutation (I97L) in human hepatitis B virus

Abstract

Mutations of human hepatitis B virus (HBV) occur frequently within the capsid (core) protein in natural infections. The most frequent mutation of the core protein in HBV from Southeast Asia occurs at amino acid 97, changing an isoleucine (I) to a leucine (L). In our systematic study of virus-host interactions, we have examined the replication efficiency of a site-directed mutant, I97L, and its parental wild-type HBV in several different hepatoma cell lines. Interestingly, we found that this capsid variant replicated in human Huh7 hepatoma cells approximately 4.8-fold better than its parental wild-type HBV. A similar phenomenon was observed in another hepatoma cell line, J3. In addition, the level of encapsidated RNA pregenome in mutant I97L was about 5.7-fold higher than that of the wild-type HBV in Huh7 cells. Unlike Huh7 cells, no significant difference in viral DNA replication between the same I97L mutant and its parental wild-type HBV was observed in HepG2, a human hepatoblastoma cell line. This finding of a profound replication advantage for mutant I97L in Huh7 and J3 cells but not in HepG2 cells may have important implications for the emergence of this mutant in chronic HBV carriers. We speculate here that the mutation confers a host factor-independent growth advantage for the survival of HBV variants in gradually dedifferentiating hepatocytes and thus helps prolong viral persistence.

FIG. 1.

Significantly increased viral DNA synthesis and virion secretion of HBV adr capsid mutant I97L in Huh7 cells but not in HepG2 cells. (a) Ten micrograms of each plasmid DNA was transfected into HepG2 and Huh7 cells. Intracellular core particles were harvested 5 days posttransfection, and the core particle-associated DNA was analyzed by Southern blot analysis with a 3.1-kb adr subtype or ayw subtype HBV probe. In comparison with wild-type HBV (WT-adr), there was at least a fourfold increase in overall DNA signal for mutant I97L in Huh7 cells, while in HepG2 cells, wild-type adr and mutant I97L did not exhibit any significant difference in replication (left panel). In contrast to subtype adr, the overall DNA signal of wild-type ayw HBV was about twofold stronger than that of the mutant F97L in both HepG2 and Huh7 cells (right panel). (b) Mutant I97L secreted more HBV virions than wild-type adr in Huh7 cells but not in HepG2 cells (left panel). This phenomenon was not observed in mutant F97L in the HBV ayw subtype (right panel). Viral particles were purified from the medium through a 20% sucrose cushion, and the HBV Dane particles were then separated by isopycnic gradient centrifugation through a 20 to 50% (wt/vol) cesium chloride gradient. Dane particles were collected according to their buoyant densities. Extracellular HBV DNA was extracted and subjected to Southern blot analysis. Full-length relaxed-circle form (RC) HBV DNA at 4.0 kb and single-stranded (SS) HBV DNA replicative intermediates at 1.5 kb are indicated by arrows.

FIG. 2.

Replication advantage phenotype of mutant I97L was observed in another hepatoma cell line, J3. Among a number of different hepatoma cell lines tested (see text for detail), only J3 was reproducibly found to behave like Huh7 cells. The Southern blot assay for viral DNA synthesis was done as described for Fig. 1a.

FIG. 3.

Mutant I97L encapsidated more pregenomic RNA than its parental wild-type virus in both HepG2 and Huh7 cells. Five days posttransfection, HepG2 and Huh7 cells were harvested, and total RNA (right panel) and core-associated RNA (left panel) were isolated. The entire amount of core-associated RNA from one 10-cm dish and 30 μg of total RNA from each sample were analyzed by Northern blot analysis with a 3.1-kb HBV double-stranded DNA probe. The difference in encapsidated RNA levels between the mutant I97L and wild-type viruses was more dramatic in Huh7 cells than in HepG2 cells. Major HBV-specific transcripts are indicated by arrows.

FIG. 4.

Comparison of steady-state levels of wild-type and mutant core proteins produced from virus replicons and protein expression vectors. Cell lysates of Huh7 cells collected 3 days posttransfection were analyzed by Western blot analysis with a rabbit anticore antibody. Plasmids SVC-WT and SVC-I97L are two core protein expression vectors of wild-type (WT) HBV and mutant I97L origins under the control of the simian virus 40 enhancer and promoter. The fact that they produced similar steady-state levels of wild-type and mutant core proteins indicated a similar degree of protein stability. Therefore, the difference in the steady-state level of core proteins expressed from the tandem dimer replicons of wild-type adr and mutant I97L reflects their difference in intracellular viral DNA replication rather than any difference in intrinsic protein stability.

FIG. 5.

Increased intracellular viral DNA synthesis of mutant I97L in Huh7 cells is likely to be caused by the acquisition of a cytosine residue at nucleotide 2191 or by the acquisition of a leucine residue at amino acid 97 of HBV core antigen. Plasmid DNAs of wild-type adr (WT-adr), mutant I97L (A2191C), or mutant I97F (A2191T) were transfected into both HepG2 and Huh7 cells. HBV core particle-associated DNA was harvested 5 days after transfection and analyzed by Southern blot analysis with an HBV double-stranded DNA probe. In Huh7 cells, mutant I97L (A2191C) but not mutant I97F (A2191T) showed increased overall HBV DNA synthesis.