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. 2013 Jul;87(14):7882-92.
doi: 10.1128/JVI.00710-13. Epub 2013 May 8.

Specific amino acid substitutions in the S protein prevent its excretion in vitro and may contribute to occult hepatitis B virus infection

Subhajit Biswas  1 , Daniel Candotti, Jean-Pierre Allain
Affiliations

Specific amino acid substitutions in the S protein prevent its excretion in vitro and may contribute to occult hepatitis B virus infection

Subhajit Biswas et al. J Virol. 2013 Jul.

Abstract

Occult hepatitis B virus (HBV) infection (OBI) is defined as low plasma level of HBV DNA with undetectable HBV surface antigen (HBsAg) outside the preseroconversion window period. The mechanisms leading to OBI remain largely unknown. The potential role of specific amino acid substitutions in the S protein from OBI in HBsAg production and excretion was examined in vitro. HBsAg was quantified in culture supernatants and cell extracts of HuH-7 cells transiently transfected with plasmids containing the S gene of eight HBsAg(+) controls and 18 OBI clones. The intracellular (IC)/extracellular (EC) HBsAg production ratio was ∼1.0 for the majority of controls. Three IC/EC HBsAg patterns were observed in OBI strains clones: pattern 1, an IC/EC ratio of 1.0, was found in 5/18 OBI clones, pattern 2, detectable IC but low or undetectable EC HBsAg (IC/EC, 7.0 to 800), was found in 6/18 OBIs, and pattern 3, low or undetectable IC and EC HBsAg, was found in 7/18 clones. Intracellular immunofluorescence staining showed that in pattern 2, HBsAg was concentrated around the nucleus, suggesting retention in the endoplasmic reticulum. The substitution M75T, Y100S, or P178R was present in 4/6 pattern 2 OBI clones. Site-directed-mutagenesis-corrected mutations reversed HBsAg excretion to pattern 1 and, when introduced into a control clone, induced pattern 2 except for Y100S. In a control and several OBIs, variants of a given quasispecies expressed HBsAg according to different patterns. However, the P178R substitution present in all cloned sequences of two OBI strains may contribute significantly to the OBI phenotype.

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Figures

Fig 1
Fig 1
Alignment of amino acid sequences deduced from cloned S genes of genotype B to D non-OBI (HBsAg+) and OBI HBV strains. Sequences of HBsAg+ and OBI clones were aligned with consensus sequences derived from 124 wild-type/HBsAg+ genotype B sequences, 95 genotype C sequences, and 150 genotype D sequences. Residues identical to the reference consensus are indicated by dots. Site-directed mutagenesis of residues in gray boxes resulted in a change in the HBsAg excretion pattern; no change was observed when residues in open boxes were corrected.
Fig 2
Fig 2
Detection of intracellular and extracellular levels of HBsAg/S protein by EIA and IFA after transfection of cloned S sequences in HuH-7 cells. (A) Average individual HBsAg production in HuH-7 cells transfected with 8 non-OBI controls and 18 OBI clones. Standard deviations are for three or more culture wells; the three different production patterns observed in OBI samples are indicated. (B) Comparison of total HBsAg production observed for the non-OBI and the three OBI patterns. Statistically significant (P < 0.05) differences determined by 1-way ANOVA/Tukey test are indicated. (C) Comparison of HBsAg IC/EC ratios observed for each control and OBI patterns. No statistical significance was observed due to three outliers with remarkably high ratios (OBI pattern 2 isolate HK01556-cl2 and non-OBI isolates M92-cl2 and M95-cl8, showing IC/EC ratios of 800, 75, and 23, respectively). (D) Relationship between IC/EC ratio and total HBsAg production. Geometric means are indicated with dashed vertical and horizontal lines; gray squares and circles identify non-OBI controls and pattern 1 OBIs with diffuse granular IF, black squares and triangles identify non-OBI controls and pattern 2 OBIs with dense aggregated IF, and open diamonds identify pattern 3 OBIs with low/undetectable HBsAg in IF.
Fig 3
Fig 3
Immunofluorescence microscopy of HuH-7 cells expressing non-OBI and OBI HBsAg. Cell nucleus were stained with DAPI (blue) and HBsAg was detected with Alexa Fluor 488-labeled mouse anti-HBsAg monoclonal IgG (green). Cells transfected with a reporter plasmid expressing LacZ were used as negative controls.
Fig 4
Fig 4
Immunofluorescence staining of native and mutated HBsAg in transfected HuH-7 cells. Amino acid substitutions introduced by site-directed mutagenesis in OBI (A) and non-OBI (B) sequences are indicated in bold.
Fig 5
Fig 5
Immunofluorescence staining of HBsAg in cells transfected with wild-type and mutated M88-cl4 sequences. The position and nature of the OBI-specific amino acid substitutions introduced into the genotype B control M88-cl4 are indicated.
Fig 6
Fig 6
Phylogenetic analysis of the S amino acid sequences of multiple clones from OBI and non-OBI strains. Phylogenetic analysis was performed as previously described (10). HBV reference sequences of genotypes/subgenotypes A1-3 and B-H are identified by their GenBank accession numbers. For clones used in HuH-7 transfection experiments, the resulting HBsAg excretion pattern is indicated by open (pattern 1), black (pattern 2) or gray (pattern 3) arrows.
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