The Mechanism of an Immature Secretion Phenotype of a Highly Frequent Naturally Occurring Missense Mutation at Codon 97 of Human Hepatitis B Virus Core Antigen

Abstract

A very frequent missense mutation at codon 97 of human hepatitis B virus (HBV) core antigen (HBcAg) has been found in chronic carriers worldwide. Functional characterization of this mutant revealed one intracellular and two extracellular phenotypes in contrast to wild-type HBV: (i) a 6- to 12-fold decrease in the level of the full-length relaxed circular DNA, a 4- to 5-fold decrease in the plus-strand DNA, and an approximately 1.8-fold decrease in the minus-strand and overall DNA levels in the intracellular viral core particles; (ii) a 5.7-fold increase in the immature secretion of Dane particles, containing minus-strand, single-stranded virion DNA; and (iii) a significant reduction of nonenveloped core particles in the medium. The steady-state levels of mutant and wild-type core proteins expressed from the same vector appeared to be similar. Using a complementation assay and gradient centrifugation analysis, we demonstrated that this mutant core protein alone is necessary and sufficient for immature secretion. The decreased level of intracellular HBV DNA is caused by both the cis defect of the mutant genome and thetrans defect of the mutant core protein. We have dissected further the relationship between the intracellular and extracellular phenotypes of mutant F97L. The pleiotropic effects of the HBcAg codon 97 mutation were observed consistently in several different experimental settings. The mechanism and biological significance of these findings are discussed.

Nucleolar Localization of Human Hepatitis B Virus Capsid Protein

“…The I97P mutant appeared with a low secretion phenotype. The I97L mutant released immature genomes as described previously (12,35,73,74). Consistent with their intracellular phenotypes shown in Fig.…”

“…Recently, members of our laboratory identified an immature secretion phenotype of a highly frequent naturally occurring HBV variant containing a leucine residue at amino acid 97 of the core protein. Unlike wild-type HBV, this 97L variant secretes almost equal amounts of mature and immature genomes (73,74). This unexpected phenomenon is not caused by instability of the core particles or by any deficiency in viral reverse transcription (35,44,73,74).…”

“…Unlike wild-type HBV, this 97L variant secretes almost equal amounts of mature and immature genomes (73,74). This unexpected phenomenon is not caused by instability of the core particles or by any deficiency in viral reverse transcription (35,44,73,74). In addition to the I97L immature secretion variant, other naturally occurring and artificially created capsid mutants exhibit a so-called low secretion phenotype with significantly reduced virion secretion while maintaining normal intracellular viral DNA replication (12,30,34,49).…”

“…Mutant 1903 bears an ablated AUG initiation codon of HBcAg in both copies of the ayw HBV tandem dimer. This mutant is thus replication defective due to the absence of core protein production (73). Mutant 1903 can be rescued to replicate if the core protein is provided by trans-complementation.…”

“…For DNA transfection, 10 g of pSVC and 10 g of mutant 1903 plasmid DNAs were adjusted to a final total of 35 g with carrier DNA and then transfected into the Huh7 human hepatoma cell line by the calcium phosphate coprecipitation method (56). The preparation of intracellular core particles and viral DNA and gradient centrifugation analyses of secreted viral particles were performed as detailed elsewhere (73).…”