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Abstract：
Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI.
摘要：
隐匿性乙型肝炎病毒感染（OBI）的特征是在不存在可检测的HBsAg的情况下存在HBVDNA。OBI是肝硬化和肝细胞癌的重要风险因子，但其发病机制尚未完全阐明。HBVpreS/S基因的突变可导致HBsAg或S蛋白的分泌受损，导致细胞中缺陷病毒或S蛋白的积累。在我们之前的工作中,M133S突变存在于OBI维持性血液透析（MHD）患者的HBVS基因中。在这项研究中,我们通过构建突变的S基因质粒，研究了S蛋白中氨基酸取代在S蛋白生产和分泌中的潜在作用，结构预测，转录组测序分析，和体外功能研究。蛋白质结构预测表明，S蛋白M133S突变体表现出亲水性修饰，膜磷脂双层内的整个结构具有更大的聚集和积累。转录组测序数据的差异基因富集分析表明，差异表达基因主要集中在内质网（ER）的蛋白质加工中。野生型和突变型组热休克家族蛋白和ER分子的表达显著增加,而线粒体相关蛋白的表达降低。免疫荧光染色和蛋白印迹显示内质网相关蛋白PDI,自噬标记LC3和溶酶体相关蛋白LAMP2与S蛋白共定位在野生型和突变株中，他们的表情增加了。线粒体相关的TOMM20蛋白也与S蛋白共表达，但在突变体中表达显著降低。S基因中的M133S突变表达为一种缺陷和错误折叠的蛋白质，该蛋白质在内质网中积累，引起分泌受损的内质网应激，这反过来触发线粒体自噬并募集溶酶体与自噬体融合，导致线粒体清除。本研究初步证明S基因中M133S的突变可引起OBI并与疾病进展有关,为OBI的诊断和治疗提供理论依据。
