Post-translational regulation of hepatitis B virus large envelope protein

项目摘要/摘要 乙肝病毒的大(L)包膜蛋白对病毒粒子的产生是必不可少的 和传染性，其细胞内高水平可导致肝癌。它有额外的preS1和preS2 结构域比小(S)包膜蛋白。S蛋白的大部分是以非传染性的方式分泌的 亚病毒颗粒(SVP)，它与诱导免疫耐受有关 慢性乙肝病毒感染的建立。L蛋白单独表达时不分泌。它会相互作用 以核心颗粒启动病毒粒子形态发生，并招募S蛋白进行病毒粒子分泌。在……里面 根据L/S的蛋白质比例，还可以抑制SVP的分泌。L蛋白质也是必不可少的 通过与高亲和力受体相互作用而启动乙肝病毒感染。尽管它很关键 在乙肝病毒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。我们之前发现通过突变来阻止S蛋白的表达 启动ATG转化为GCG取消了M蛋白，表明其稳定性依赖于S蛋白。 尽管细胞分泌受阻，但细胞内L蛋白水平也明显降低。非常 最近，我们发现这个特殊的突变体可以产生一个矮小的不分泌的S 蛋白质通过翻译起始于下游的ATG。进一步将该ATG突变为GCG 使细胞内L蛋白降低到极低水平。然而，突变了第一和第二个 S基因中的框内ATG密码子引入S结构域的氨基酸替换 L蛋白。为了验证L蛋白水平的降低是由于S蛋白表达的缺失 比起L蛋白的突变，在目标1中我们将测试S基因的ATG密码子突变成 其他密码子，如ATA、AAG和TTG。前S1区含有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 取消S蛋白表达。是否也在转录水平上失去了S蛋白的表达 细胞内缩短型L蛋白的水平将被测定。目标2将确定该角色 核心蛋白在维持L蛋白水平方面的作用，并检查其与S蛋白的协同作用。我们的初步研究 提示核心蛋白的表达是维持细胞内和细胞外的必需的。 L蛋白水平。在目标3中，我们将验证细胞内L蛋白水平的大幅降低是否 归因于加速降解，并检查在反式中提供S或核心蛋白是否可以挽救 L蛋白水平。我们推测，在乙肝病毒粒子形态发生过程中，L蛋白通过 它与核心颗粒或与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蛋白质水平的新机制，应该有 治疗方面的影响。

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.