Data_Sheet_2_E2 Site Mutations in S Protein Strongly Affect Hepatitis B Surface Antigen Detection in the Occult Hepatitis B Virus.PDF

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.

隐匿性乙型肝炎病毒感染（occult hepatitis B infection, OBI）的发病机制尚未完全阐明。我们既往的研究发现，S蛋白内存在与OBI相关的新型突变E2G，该突变可导致乙型肝炎表面抗原（hepatitis B surface antigen, HBsAg）分泌障碍，进而引发B基因型OBI病例出现胞内抗原蓄积。为进一步探讨E2位点突变在OBI发生中的作用，本研究对119株从献血者中分离得到的OBI毒株进行了该位点突变分析；同时以109株野生型HBV毒株（HBsAg阳性/HBV DNA阳性）作为对照组。此外，为验证E2位点突变的功能效应，我们构建了HBV B、C基因型的保守型1.3倍全长基因表达载体（pHBV1.3B与pHBV1.3C）。随后，分别以pHBV1.3B或pHBV1.3C为骨架构建E2突变体质粒，并转染至HepG2细胞中。采用电化学发光法与细胞免疫组化法分析细胞外与细胞内的HBsAg水平；利用相关生物信息学软件分析携带或不携带E2突变的S蛋白的结构特征。在119株OBI毒株中，21.8%（26/119）存在E2突变（E2G/A/V/D），而对照组未检出任何E2突变。E2G/A/V/D突变可显著影响HBsAg的胞外与胞内水平（p < 0.05）。值得注意的是，与B基因型E2G突变可导致HBsAg胞内蓄积与分泌减少（p < 0.05）不同，C基因型E2G突变可同时显著降低胞外（相较于pHBV1.3C组，仅为0.46%）与胞内（相较于pHBV1.3C组，仅为11.2%）的HBsAg水平（p < 0.05）。与此同时，针对E2G/A突变，其相对胞内HBsAg水平（为胞外水平的110.7%~338.3%）与荧光强度（相较于基因型匹配的pHBV1.3B/C组，为1.5~2.4倍）均显著升高（p < 0.05）。进一步研究发现，仅在存在分泌缺陷的E2G/A突变型S蛋白中，可检测到位于27、28位氨基酸的典型肽酶切割位点的N端信号肽序列。本研究结果提示：（1）E2G/A/V/D突变可显著影响HBsAg的检测；（2）E2G突变导致OBI的潜在机制在B基因型与C基因型毒株中存在显著差异；（3）E2G/A突变可产生N端截短型S蛋白，这可能是OBI病例中HBsAg分泌障碍的原因。