ABSTRACT

Occult HBV infection (OBI) is a special infection status during Hepatitis B virus (HBV) infection. The underlying mechanism of its occurrence remains unclear. This study conducted sequencing analysis on 104 OBI plasma samples and 524 HBsAg positive samples from 29 blood centres, and searched for high-frequency mutations in transmembrane domain (TMD) of S protein in the OBI population. Plasmids with TMD high-frequency mutations were constructed, in vivo and in vitro functional experiments were performed to investigate possible molecular mechanisms of OBI occurrence. We found 22 high-frequency TMD mutations in genotype B OBI strains. Among them, five mutations can lead to impairment of HBsAg secretion; seven mutations had accumulated intracellular HBsAg while extracellular HBsAg didn’t decrease compared to wildtype. This study chose C85R from TMD2, F220C, and F220Y from TMD4 for further exploration. Protein structure predication showed these three mutant HBsAg displayed changed hydrophilic properties and tended to accumulate in the phospholipid bilayer of cell membrane. Mutant HBsAg’s secretion disorder may induce OBI. On the other hand, V168A + V177A from TMD3 expressed increased HBsAg both in intracellular and extracellular levels. This mutation had most unstable natural conformation and may be inclined to transition into V177A or V168A + S174N + V177A. These three mutations were more prone to mixed infection, presenting a state of coexistence, thus approaching the impaired secretion pattern of OBI. This study demonstrated TMD mutations could contribute to the occurrence of OBI and provided a theoretical basis for OBI study and the functional cure of chronic hepatitis B virus infection.

摘要

隐匿性HBV感染（OBI）是乙型肝炎病毒（HBV）感染过程中的一种特殊感染状态。其发生的潜在机制仍不清楚。本研究对来自 29 个血液中心的 104 份 OBI 血浆样本和 524 份 HBsAg 阳性样本进行测序分析，并在 OBI 人群中寻找 S 蛋白跨膜结构域 (TMD) 的高频突变。在体内和体外构建了具有 TMD 高频突变的质粒进行了功能实验以研究 OBI 发生的可能分子机制。我们在基因型 B OBI 菌株中发现了 22 个高频 TMD 突变。其中，5个突变可导致HBsAg分泌受损；与野生型相比，7 个突变积累了细胞内 HBsAg，而细胞外 HBsAg 没有减少。本研究选择TMD2中的C85R、TMD4中的F220C和F220Y进行进一步探索。蛋白质结构预测表明这三个突变HBsAg表现出改变的亲水特性，并倾向于在细胞膜的磷脂双层中积累。突变HBsAg的分泌障碍可能诱发OBI。另一方面，来自 TMD3 的 V168A + V177A 在细胞内和细胞外水平均表达增加的 HBsAg。这种突变具有最不稳定的自然构象，可能倾向于转变为 V177A 或 V168A + S174N + V177A。这三个突变更容易发生混合感染，呈现共存状态，从而接近OBI受损的分泌模式。本研究表明TMD突变可能导致OBI的发生，为OBI研究和慢性乙型肝炎病毒感染的功能性治愈提供了理论依据。