Post-translational regulation of hepatitis B virus large envelope protein

项目概要/摘要 乙型肝炎病毒 (HBV) 的大 (L) 包膜蛋白对于病毒粒子的产生至关重要 并具有传染性，其细胞内含量高可引起肝癌。它有额外的 preS1 preS2 结构域比小 (S) 包膜蛋白大。大部分 S 蛋白以非感染性形式分泌 亚病毒颗粒（SVP），与诱导免疫耐受有关 慢性乙型肝炎病毒感染的建立。 L蛋白单独表达时不分泌。它相互作用 与核心颗粒启动病毒颗粒形态发生，并招募 S 蛋白进行病毒颗粒分泌。在 这样做还可以根据 L/S 蛋白比率抑制 SVP 分泌。 L蛋白也是必不可少的 通过与高亲和力受体相互作用启动 HBV 感染。尽管它很关键 在 HBV 生物学和发病机制中的重要性，但对其细胞内水平的调节知之甚少 在翻译后步骤。我们之前发现通过突变来阻止S蛋白表达 起始 ATG 进入 GCG 消除了 M 蛋白，表明其稳定性依赖于 S 蛋白。 尽管分泌受阻，L蛋白的细胞内水平也显着降低。非常 最近，我们发现这种特殊的突变体可以产生缩短且非分泌的 S 蛋白质通过下游 ATG 的翻译起始。进一步将 ATG 突变为 GCG 细胞内L蛋白减少至极低水平。尽管如此，改变第一个和第二个 S基因中的框内ATG密码子进入GCG，在S结构域中引入氨基酸取代 L蛋白。验证 L 蛋白水平降低是由于 S 蛋白表达丢失而不是 与 L 蛋白的突变相比，在目标 1 中，我们将测试 S 基因 ATG 密码子突变为的影响 其他密码子，例如 ATA、AAG 和 TTG。 preS1 区域包含 2.1-kb RNA 的启动子 对于 M/S 蛋白，我们最近发现了许多自然发生的框内 preS1 缺失 废除S蛋白表达。转录水平上是否也失去了S蛋白表达 将测定缩短的L蛋白的细胞内水平的降低。目标 2 将建立角色 核心蛋白在维持L蛋白水平中的作用，并检查其与S蛋白的协同作用。我们的试点研究 表明需要核心蛋白表达来维持细胞内和细胞外 L蛋白水平。在目标 3 中，我们将验证细胞内 L 蛋白水平大幅降低是否是 归因于加速降解，并检查提供反式 S 或核心蛋白是否可以挽救 L蛋白水平。我们假设在 HBV 病毒粒子形态发生过程中，L 蛋白通过 它与核心颗粒或S蛋白的分子相互作用。拟议的研究将揭示 在翻译后水平控制L蛋白水平的新机制，应该具有 治疗意义。

Project Summary/Abstract The large (L) envelope protein of hepatitis B virus (HBV) is essential for virion production and infectivity, and its high intracellular level can cause liver cancer. It has extra preS1 + preS2 domains than small (S) envelope protein. Majority of the S protein is secreted as noninfectious subviral particles (SVPs), which have been implicated in the induction of immune tolerance for establishment of chronic HBV infection. L protein is not secreted when expressed alone. It interacts with core particle to initiate virion morphogenesis, and recruits the S protein for virion secretion. In doing so it also suppresses SVP secretion according to L/S protein ratio. L protein is also essential for initiation of HBV infection by interacting with the high-affinity receptor. Despite its critical importance in HBV biology and pathogenesis, little is known about regulation of its intracellular level at the post-translational step. We previously found that preventing S protein expression by mutating the initiating ATG into GCG abolished M protein, suggesting its reliance on S protein for stability. The intracellular level of L protein was also markedly reduced despite blocked secretion. Very recently, we discovered this particular mutant could produce a shortened and non-secreted S protein through translation initiation from a downstream ATG. Further mutating that ATG into GCG diminished intracellular L protein to extremely low level. Nevertheless, mutating the first and second in-frame ATG codons in the S gene into GCG introduces amino acid substitutions in the S domain of L protein. To verify that reduced L protein level is attributed to lost S protein expression rather than mutations in L protein, in Aim 1 we will test the impact of mutating the S gene ATG codon into other codons such as ATA, AAG, and TTG. The preS1 region harbors the promoter for 2.1-kb RNA for M/S proteins, and we recently found many naturally occurring in-frame preS1 deletions abolished S protein expression. Whether lost S protein expression at the transcriptional level also diminishes intracellular level of shortened L protein will be determined. Aim 2 will establish the role of core protein in sustaining L protein level and check for its synergy with S protein. Our pilot study suggested that core protein expression was required to sustain both intracellular and extracellular levels of L protein. In Aim 3, we will verify whether much reduced intracellular level of L protein is attributed to accelerated degradation, and examine if providing S or core protein in trans can rescue the L protein level. We hypothesize that during HBV virion morphogenesis, L protein is stabilized by its molecular interaction with core particles or with S protein. The proposed studies will reveal a novel mechanism to control L protein level at the post-translational level, which should have therapeutic implications.