Abstract
The mechanism of occult hepatitis B infection (OBI) has not yet been fully clarified. Our previous research found that novel OBI-related mutation within S protein, E2G, could cause the hepatitis B surface antigen (HBsAg) secretion impairment, which resulted in intracellular accumulation in OBI of genotype B. Here, to further explore the role of E2 site mutations in the occurrence of OBI, we analyzed these site mutations among 119 OBI strains identified from blood donors. Meanwhile, 109 wild-type HBV strains (HBsAg positive/HBV DNA positive) were used as control group. Furthermore, to verify the E2 site mutations, two conservative 1.3-fold full-gene expression vectors of HBV genotype B and C (pHBV1.3B and pHBV1.3C) were constructed. Then, the E2 mutant plasmids on the basis of pHBV1.3B or pHBV1.3C were constructed and transfected into HepG2 cells, respectively. The extracellular and intracellular HBsAg were analyzed by electrochemical luminescence and cellular immunohistochemistry. The structural characteristics of S proteins with or without E2 mutations were analyzed using relevant bioinformatics software. E2 mutations (E2G/A/V/D) existed in 21.8% (26/119) of OBIs, while no E2 mutations were found in the control group. E2G/A/V/D mutations could strongly affect extracellular and intracellular level of HBsAg (p < 0.05). Notably, unlike E2G in genotype B that could cause HBsAg intracellular accumulation and secretion decrease (p < 0.05), E2G in genotype C could lead to a very significant HBsAg decrease both extracellularly (0.46% vs. pHBV1.3C) and intracellularly (11.2% vs. pHBV1.3C) (p < 0.05). Meanwhile, for E2G/A mutations, the relative intracellular HBsAg (110.7–338.3% vs. extracellular) and its fluorescence intensity (1.5–2.4-fold vs. with genotype-matched pHBV1.3B/C) were significantly higher (p < 0.05). Furthermore, N-terminal signal peptides, with a typical cleavage site for peptidase at positions 27 and 28, were exclusively detected in S proteins with secretion-defective mutants (E2G/A). Our findings suggest that: (1) E2G/A/V/D mutations were confirmed to significantly influence the detection of HBsAg, (2) the underlying mechanism of OBI caused by E2G mutation is quite different between genotype B and genotype C, and (3) E2G/A could produce a N-terminal truncated S protein, which might attribute to the HBsAg secretion impairment in the OBIs.
Highlights
MATERIALS AND METHODSHepatitis B virus (HBV) infection is a global health problem
Mutation E2G/A/V/D were identified in 1.1–11.2% of the genotype B Occult HBV infection (OBI), and E2G/A/D were identified in 3.3– 13.3% of the genotype C OBIs (Table 1)
This study investigated a series of S protein E2 mutations in OBI genotypes B and C, which play an important role in the development of OBI
Summary
Hepatitis B virus (HBV) infection is a global health problem. The pre-S1/S2/S encodes the three hepatitis B surface antigen (HBsAg) proteins, termed large, middle, and small surface antigen protein (Lamontagne et al, 2016). The small surface antigen (S protein) is the predominant HBsAg protein, which consists of 226 amino acids (aa) and encoded by S gene (nt155832). HBsAg is an important serological biomarker for the detection of HBV, which is present on the surface of 22 nm subviral particles (SVPs) and 42 nm virions. The SVPs appear as spheres or filaments composed of empty envelopes and are secreted 103-fold to 106fold excess over virions (Hu and Liu, 2017), HBsAg is virtually equivalent to SVPs. SVPs are synthesized at the rough endoplasmic reticulum (ER) and secreted via the constitutive secretory pathway of the host cell (Watanabe et al, 2007; Jiang et al, 2015)
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