Post-translational regulation of hepatitis B virus large envelope protein

项目概要/摘要 B型肝炎病毒（HBV）的大（L）包膜蛋白是病毒体产生所必需的 和感染性，其高细胞内水平可导致肝癌。它有额外的preS 1 + preS 2 结构域比小（S）包膜蛋白。大部分S蛋白以非感染性形式分泌 亚病毒颗粒（SVP），其已经涉及对以下的免疫耐受的诱导： 建立慢性HBV感染。L蛋白单独表达时不分泌。其交互 具有核心颗粒以启动病毒体形态发生，并募集S蛋白用于病毒体分泌。在 这样，它还根据L/S蛋白质比率抑制SVP分泌。L蛋白质也是必不可少的 通过与高亲和力受体相互作用启动HBV感染。尽管其关键 在HBV生物学和发病机制中的重要性，对其细胞内水平的调节知之甚少 在翻译后阶段。我们以前发现，通过突变阻止S蛋白表达， 起始ATG转化为GCG后，M蛋白消失，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著降低。非常 最近，我们发现这种特殊的突变体可以产生一个缩短的和非分泌的S 蛋白质通过下游ATG的翻译起始。将ATG进一步突变为GCG 使细胞内L蛋白降至极低水平。然而，第一个和第二个突变 将S基因中的符合读框的ATG密码子引入GCG中， L蛋白质。为了验证L蛋白水平的降低归因于S蛋白表达的丧失，而不是 相比于L蛋白中的突变，在目标1中，我们将测试将S基因ATG密码子突变为 其他密码子如ATA、AAG和TTG。preS 1区含有2.1-kb RNA的启动子 对于M/S蛋白，我们最近发现许多自然发生的框内preS 1缺失 抑制S蛋白表达。是否在转录水平上失去了S蛋白的表达， 减少缩短的L蛋白的细胞内水平。目标2将确立角色 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 表明核心蛋白表达是维持细胞内和细胞外 L蛋白水平。在目标3中，我们将验证是否大量降低的L蛋白的细胞内水平， 归因于加速降解，并检查是否提供S或反式核心蛋白可以拯救 L蛋白水平。我们假设在HBV病毒粒子形态发生期间，L蛋白通过以下方式稳定 其与核心颗粒或S蛋白的分子相互作用。这些研究将揭示一个 在翻译后水平上控制L蛋白水平的新机制， 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.