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.Human hepatitis B virus (HBV) can cause acute and chronic hepatitis in humans, with the latter often resulting in cirrhosis and hepatocellular carcinoma (6,11,55,56). HBV is an enveloped virus, which consists of an outer lipoprotein envelope and an inner nucleocapsid containing a 3.2-kb partially doublestranded DNA genome. The nucleocapsid, formed by a 183-amino-acid core antigen, assembles in the cytoplasm with a 3.2-kb pregenomic RNA and the viral polymerase. The encapsidated pregenomic RNA is then retrotranscribed into viral DNA (20,53).Because the polymerase of HBV lacks a proofreading function, HBV has a low fidelity in replication and thus tends to produce sequence variants at a high frequency. Naturally occurring mutations in HBV have been hypothesized to play a role in the pathology of HBV-related diseases and in the persistence of HBV infection (23, 27, 59, 60, 68, 69). As a major T-cell target (13, 42), the core protein accumulates frequent mutations in chronic carrier patients with active liver disease (1,5,8,14,15,16,27).Within the core protein, the most frequent mutation occurs at amino acid 97 (1,5,14,15,16,17,21,22,27,29,33,40,41,43,48,50,61,64,65,70). The codon 97 mutation changes the wild-type amino acid from a phenylalanine to a leucine in the ayw subtype (F97L) or from an isoleucine to a leucine in the adr subtype (I97L). Previously, we identified an "immature secretion" phenotype for capsid variant 97L (referring to both I97L and F97L), which is characterized by secretion of an excessive amount of Dane particles containing immature singlestranded DNA intermediates in both the ayw and adr subtypes (70, 71). ...

Stability and Morphology Comparisons of Self-AssembledVirus-Like Particles from Wild-Type and Mutant Human Hepatitis B VirusCapsidProteins

“…1B). To confirm the identity of the expressed and purified HBcAg, we performed immunoblot analysis with a rabbit polyclonal anti-core antibody (27). Both monomeric and dimeric HBcAg1-149 can be detected by immunological crossreactivity using Western blot analysis (right panel, Fig.…”

“…Despite the prevalence of this mutation reported by many different research groups (see references in Suk et al [27]), the functional significance of this hotspot mutation remains to be investigated. Previously, we noted two distinct phenotypes associated with this core mutation.…”

“…This phenotype represents an interesting exception to the dogma of preferential secretion of virions containing mature genomes (partially double-stranded relaxed circle DNA) (11,21,28,30). Second, a replication advantage and host factorindependent phenotype was found to be associated with the mutant I97L in the human hepatoma cell line Huh7, but not in a hepatoblastoma cell line HepG2 (27). To date, the structural basis for either the immature secretion or replication advantage phenotypes has remained unclear.…”

“…Encapsidated nucleic acids were stained with EtBr, and the same gel was subsequently stained with protein-specific Coomassie blue. A rabbit polyclonal anticore antibody was used to detect the HBcAg (27).…”

Hepatitis B Virus Capsid Assembly Is Enhanced by Naturally Occurring Mutation F97L

“…Mutation of amino acid 97 to leucine, one of the most common mutations of the core protein seen in chronic infections (13), has been found to affect nucleic acid metabolism (26,(35)(36)(37). Depending on the strain of HBV, residue 97 is phenylalanine (e.g., ayw and adyw) or isoleucine (e.g., adr and adw).…”