Effects of amino acid substitutions in hepatitis B virus surface protein on virion secretion, antigenicity, HBsAg and viral DNA

Abstract

Background & aims: As important virological markers, serum hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels show large fluctuations among chronic hepatitis B patients. The aim of this study was to reveal the potential impact and mechanisms of amino acid substitutions in small hepatitis B surface proteins (SHBs) on serum HBsAg and HBV DNA levels.

Methods: Serum samples from 230 untreated chronic hepatitis B patients with genotype C HBV were analyzed in terms of HBV DNA levels, serological markers of HBV infection and SHBs sequences. In vitro functional analysis of the identified SHBs mutants was performed.

Results: Among 230 SHBs sequences, there were 39 (16.96%) sequences with no mutation detected (wild-type) and 191 (83.04%) with single or multiple mutations. SHBs consist of 226 amino acids, of which 104 (46.02%) had mutations in our study. Some mutations (e.g., sE2G, sL21S, sR24K, sT47A/K, sC69stop (sC69∗), sL95W, sL98V, and sG145R) negatively correlated with serum HBsAg levels. HBsAg and HBV DNA levels from this group of patients had a positive correlation (r=0.61, p<0.001). In vitro analysis showed that these mutations reduced extracellular HBsAg and HBV DNA levels by restricting virion secretion and antibody binding capacity. Virion secretion could be rescued for sE2G, sC69∗, and sG145R by co-expression of wild-type HBsAg.

Conclusion: The serum HBsAg levels were lower in untreated CHB patients with novel SHBs mutations outside the major antigenic region than those without mutations. Underlying mechanisms include impairment of virion secretion and lower binding affinity to antibodies used for HBsAg measurements.

Lay summary: The hepatitis B surface antigen (HBsAg) is a major viral protein of the hepatitis B virus (HBV) secreted into patient blood serum and its quantification value serves as an important marker for the evaluation of chronic HBV infection and antiviral response. We found a few new amino acid substitutions in HBsAg associated with lower serum HBsAg and HBV DNA levels. These different substitutions might impair virion secretion, change the ability of HBsAg to bind to antibodies, or impact HBV replication. These could all result in decreased detectable levels of serum HBsAg. The factors affecting circulating HBsAg level and HBsAg detection are varied and caution is needed when interpreting clinical significance of serum HBsAg levels. Clinical trial number: NCT01088009.

Keywords: Amino acid substitution; Antigenicity; DNA; HBV; HBsAg mutation; Hepatitis B surface antigens; Membrane proteins; Viral; Virion secretion; ‘a’ determinant.

Conflict of interest statement

Conflict of interest: none of the authors has any conflict of interest.

Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Figures

Fig. 1. Mutational profile of SHBs among 230 CHB patients with genotype C HBV infection

(A) Map of amino acid substitution frequencies at a total of 226 SHBs sites [9]. The lower schematic highlights functional regions of SHBs and three HBV envelope proteins (LHBs, MHBs and SHBs) share a common C terminus. (B) Comparison of HBsAg levels between WT and mutant groups stratified by the median in 230 samples. Dots represent individual values and horizontal lines as median value with interquartile range. (C) The identified individual mutations occurred mostly in the Low HBsAg group (p < 0.05). The group Low and High was stratified by the median HBsAg level of 4.10 log10 IU/ml in 191 patients harboring SHBs substitutions. (D) Comparison of HBsAg levels between the identified mutants and WT. Boxes represent values and bars as median with range. AA: amino acid; CHB: chronic hepatitis B; SHBs: small hepatitits B surface protein; MHBs: middle hepatitis B surface protein; LHBs: large hepatitis B surface protein; TM: transmembrane; MHR: major hydrophilic region; CTL: cytotoxic T lymphocytes; WT, wild-type. ANOVA test (B and D) and Fisher’s exact test (C) were employed.

Fig. 2. SHBs mutations affect serum HBsAg and HBV DNA levels in HBeAg-positive CHB patients

Correlation of HBsAg and HBV DNA levels in (A) whole, (B) mutants groups of the samples. Comparison of (C) HBsAg levels and (D) HBV DNA levels among wild type, mutants and single mutation groups. Mutants group indicates that the sequences with the selected mutations are grouped. Single mutation group indicates that the sequences with only one mutation occurred from the selected mutation sequences are grouped. Dots represent individual values and horizontal lines as median value with interquartile range. ***, p < 0.0001; **, p < 0.001; *, p < 0.05. Spearman rank correlation test and ANOVA test were employed.

Fig. 3. Comparison of WT and mutant HBsAg expression in transfected HepG2 cells

WT and the indicated mutant derivatives were constructed on the pBB4.5 1.2/PC HBV background and used to transfect HepG2 cells. Supernatant and cells were harvest 72 hours post transfection. (A) Extracellular and (B) Intracellular HBsAg levels were measured by ELISA and Western blot with (C) polyclonal anti-HBs and (D) monoclonal anti-pre-S2 antibody. *, p < 0.05. Student’s T test was employed.

Fig. 4. Comparison of HBsAg expression of HBV WT and mutants fused with HA-tag in transfected HepG2 cells

To abolish the influence of HBsAg antigenicity, a HA-tag was fused to S protein for detection with Western blot. (A) Scheme of construction of HBV S gene with HA-tag. (B) Western blot was performed to detect extracellular and intracellular WT and mutant HBsAg levels using anti-HA antibody.

Fig. 5. Comparison of HBV replication between HBV WT and mutants in transfected HepG2 cells

(A) The concomitant AA mutations in SHBs and RT. Analysis of (B) extracellular HBsAg levels, (C) extracellular, (D) intracellular HBV DNA levels, (E) total mRNA, (F) pgRNA, (G) total mRNA in YMHD and (H) pgRNA in YMHD of HBV WT and mutants 72 hours after transfecting HepG2 cells. YMHD RT mutation was used as a control for input plasmid DNA background. Data shown as fold change relative to WT. *, p < 0.05. Student’s T test was employed.

Fig. 6. Co-expression of WT HBsAg and mutant HBV plasmids restores virion secretion

ELISA analysis of extracellular HBsAg and qPCR analysis of extracellular HBV DNA upon co-transfection of WT or mutant replication constructs together with HBsAg expressing plasmid pLMS at different ratios. (A and B) at a 5:1 ratio of replication construct to pLMS (500 ng to 100 ng), (C and D) at a 1:1 ratio (500 ng to 500 ng) and (E and F) at a 1:5 ratio (100 ng to 500 ng). The pBluescript II KS (+) plasmid was used as a control (Mock). **, p < 0.01; *, p < 0.05. Student’s T test was employed.