A frequent, naturally occurring mutation (P130T) of human hepatitis B virus core antigen is compensatory for immature secretion phenotype of another frequent variant (I97L)

Abstract

A frequent mutation at codon 97 of human hepatitis B virus core antigen has been shown to cause an "immature secretion" phenotype, featuring nonselective and excessive secretions of virions containing immature viral genome. Our current study demonstrates that this abnormality can be efficiently offset by another frequent core mutation, P130T.

FIG. 1

The intracellular hypermaturation phenotype of mutation P130T displays an increased relative amount of intracellular near-full-length RC-form DNA. Ten micrograms of plasmid DNA was adjusted to a total of 35 μg of DNA with a carrier and transfected to human hepatoma cell line HepG2. Core-associated HBV DNA was purified 7 days posttransfection as described previously (33). HBV DNA replication intermediates were separated by gel electrophoresis and detected by Southern blot analysis with a 3.2-kb HBV (adr) full-length probe. Characteristic HBV DNA replication intermediates are indicated by arrows. WT, wild type; SS, ssDNA.

FIG. 2

Mutation P130T can reverse the immature secretion phenotype of mutation I97L. (A) Secreted HBV particles were analyzed by CsCl gradient centrifugation. The media were collected on days 5 and 7 posttransfection. Virus particles were then purified through a 20% sucrose cushion and subjected to isopycnic centrifugation in a gradient of 20 to 50% (wt/vol) cesium chloride. Fractions were separated according to their buoyant density and then submitted to assays for HBsAg (▴) (Abbott Auszyme EIA [enzyme immunoassay] kit) and HBeAg (■) (Abbott HBe rDNA [recombinant DNA] EIA kit). The enveloped virions (HBsAg positive and HBcAg negative) band at a density near 1.24 g/cm³ around fractions 10 to 14. The nonenveloped core particles (HBsAg negative and HBcAg positive) band at a density near 1.35 g/cm³ around fractions 2 to 6. WT, wild type. (B) Extracellular HBV DNA was analyzed by Southern blotting. Extracellular HBV DNA was purified and collected into either the core (pooled from fractions 2, 4, and 6) or Dane (pooled from fractions 10, 12, and 14) particle fractions. HBV-specific signal was detected by Southern blot assay as described above. C, nonenveloped core particles; D, enveloped Dane particles; SS, ssDNA.

FIG. 2

Mutation P130T can reverse the immature secretion phenotype of mutation I97L. (A) Secreted HBV particles were analyzed by CsCl gradient centrifugation. The media were collected on days 5 and 7 posttransfection. Virus particles were then purified through a 20% sucrose cushion and subjected to isopycnic centrifugation in a gradient of 20 to 50% (wt/vol) cesium chloride. Fractions were separated according to their buoyant density and then submitted to assays for HBsAg (▴) (Abbott Auszyme EIA [enzyme immunoassay] kit) and HBeAg (■) (Abbott HBe rDNA [recombinant DNA] EIA kit). The enveloped virions (HBsAg positive and HBcAg negative) band at a density near 1.24 g/cm³ around fractions 10 to 14. The nonenveloped core particles (HBsAg negative and HBcAg positive) band at a density near 1.35 g/cm³ around fractions 2 to 6. WT, wild type. (B) Extracellular HBV DNA was analyzed by Southern blotting. Extracellular HBV DNA was purified and collected into either the core (pooled from fractions 2, 4, and 6) or Dane (pooled from fractions 10, 12, and 14) particle fractions. HBV-specific signal was detected by Southern blot assay as described above. C, nonenveloped core particles; D, enveloped Dane particles; SS, ssDNA.