揭示乙型肝炎表面抗原新突变对病毒复制的影响及突变株传播的新途径

Hepatitis B surface protein/antigen (HBs/HBsAg) is an important component of hepatitis B virus (HBV) and a crucial serological marker of HBV infection. However, the abundant HBs mutants are known to exist in patients with chronic HBV infection and have virological and clinical implications. We previously found that some amino acid (AA) mutations occurring outside the antigenic determinant region (“a” determinant) of HBs sequences could significantly affect viral and clinical characteristics. The mutations outside “a” determinant have never been the study focus probably due to their seemingly unimportant structural locations in HBs. Lack of efficient infectious experimental system to study mutation impacts on viral transmission might be another reason before the discovery of HBV cellular receptor (sodium taurocholate cotransporting polypeptide, NTCP). In this study we will study the impacts of some of the novel AA mutations occurring outside “a” determinant on viral replication, secretion and antigenicity of HBsAg. In addition, we will study if some of these mutants with secretion deficiency could spread by a cell-to-cell transmission (CCT) mechanism. CCT has been a yet unrevealed viral spread pathway in HBV, thought it has been shown important for human immunodeficiency virus and hepatitis C virus survival, transmission and antiviral resistance. Our preliminary studies identified about 10 novel HBs AA mutations (e.g. sE2G、sN3S/T and sS53L), which were from HBs sequences obtained from untreated and hepatitis B e antigen positive patients with chronic infection of HBV genotype C. These mutations have been associated with low levels of serum HBsAg, or have very high mutation detection rates (>10%). All newly found AA mutations are located outside “a” determinant. We have characterized some of them and got to know the novel HBs mutations might affect viral replication and secretion. In this study, we will continue to finish the constructions up to 10 mutant clones competent for HBV replication. Then, we will use a serial of hepatocellular carcinoma cell lines (HepG2, HepG2-GFP, HepG2-NTCP-GFP, HepG2-NTCP and so on) and different co-cultures of them, and primary hepatocytes as in vitro transfection or infection experimental systems to clarify the impacts of these novel HBs mutations on HBV replication, secretion, antigenicity and transmission. Most of these cell lines are available in our laboratory, for those yet unavailable; we will establish them by techniques such as CRISPR/Cas9. Hopefully, we would demonstrate that HBV or some of HBs novel mutants could spread by CCT pathway. Further, we will study if NTCP and some other cell surface molecules, such as claudin and apolipoprotein, might mediate CCT in HBV. The updated knowledge will help us to better interpret the clinical implications of serum HBsAg level, to design more sensitive diagnostic reagents, and to develop anti-HBV drugs targeting the novel CCT pathway.

乙型肝炎表面蛋白/抗原（HBs/HBsAg）是HBV的重要成分和重要诊疗血清标志物。课题以既往很少关注的HBs抗原决定簇之外的氨基酸突变为研究对象，研究新突变对HBV影响；利用分泌有缺陷突变株，研究HBV是否可通过细胞间直接传播（CCT）途径播散。课题以未经抗病毒治疗的C基因型感染者来源的HBs序列分析为基础，鉴定出与低值血清HBsAg水平有关、或突变率很高的10种HBs新突变（如sE2G）。构建复制型突变质粒，借助肝癌细胞系、过表达HBV细胞受体NTCP的细胞系、原代肝细胞、有绿色荧光蛋白标记的感染细胞-未感染细胞共培养体系等，在转染或感染实验中研究新突变对病毒复制、分泌和传播的影响，揭示其存在CCT传播新途径，再研究NTCP、claudin和载脂蛋白等是否介导CCT。结果为准确解读血清HBsAg水平意义、研发高灵敏度诊断试剂和以CCT为靶点新型抗病毒药物提供重要理论依据。

最先与肝细胞发生接触的乙型肝炎（乙肝）病毒（HBV）的分子是乙肝表面蛋白（HBs），包括大中小HBs（L/M/SHBs），它们在HBV感染的建立、复制和细胞间传播中分工合作，具有重要生物学功能。HBs易突变，有些突变具有重要的生物学和临床影响。本研究发现了HBs新突变；利用肝癌细胞系（HepG2-NTCP、HepG2、HepG2-NTCP-GFP和HepG2-GFP等）在体外研究了重要新突变的特性。主要发现有：（1）鉴定出与高水平血清乙肝表面抗原（HBsAg）相关的SHBs新突变sC76Y和sI218L，它们不在SHBs抗原决定簇内，但可通过不同机制影响HBV复制和分泌；（2）鉴定出SHBs新突变sC69*，相关毒株分泌和感染性差，靠准种中野生型HBs包装生存；它对野生型准种的回馈可能是更强的抑制天然免疫应答的能力，有助于准种群体生存和持续感染；（3）发现主突变sI126T对HBsAg的抗原性和分泌性的影响，受到SHBs其他位点氨基酸联合突变的影响，这种基因上位效应首次在HBV中得到证实；（4）证实LHBs的preS1的N末端异质性序列对HBV感染性的影响具有基因型特异性，基因D型感染性最强，B和C型差；（5）初步发现HBV可能存在细胞间直接传播（CCT），但夯实证据需要更好的体外感染体系，为此我们建立了通过添加干细胞专用基质胶（M-hq）来提高HBV对HepG2-NTCP细胞感染率的新的细胞感染体系，在此体系中证实肝细胞表面钠离子-牛磺胆酸共转运蛋白（NTCP）和表皮生长因子受体（EGFR）对病毒感染入胞很重要，显示了此体系的可应用性。本研究发现和构建的特性各异的多种HBs突变株、感染率高的新的细胞体系和适合HBV胞间传播研究的实验体系，为未来的研究留下了丰富的实验材料资源和宝贵的技术平台。对HBs突变株生物学特性、生存和传播机制的揭示，为理解病毒突变的生物学和临床意义奠定了基础，为血清HBsAg水平的个体化解读和优化诊断试剂提供了理论依据。研究成果发表了8篇SCI论文，课题组研究生2位获得博士学位，3位获得硕士学位。

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