Control of HBV replication at the step of core and P protein translation

描述(申请人提供)：乙肝病毒(乙肝病毒)，它会导致肝硬变和肝细胞癌，有一个小的，紧凑的基因组。基因组复制所需的唯一转录本是3.5kb的前基因组(PG)RNA。它既是核心蛋白和P蛋白翻译的双顺反子，也是被P蛋白包裹并转化为双链DNA的基因组前体。由于衣壳是由240个拷贝的核心蛋白组装而成，但包装的可能只有一个P蛋白分子，因此有效的基因组复制需要适当的核心/P蛋白翻译比例。P蛋白翻译涉及到 核糖体泄漏扫描上游的AUG密码子，包括核心基因的密码子，但控制机制仍然不清楚。G基因在其核心基因5‘端有一个独特的36个核苷酸插入，无论是在G基因中还是在人工引入其他基因类型时，它都显著地增强了核心蛋白的翻译。这种核心蛋白翻译的增加更多地与非G基因型的基因组复制受损有关，最可能的原因是P蛋白翻译的相应减少。矛盾的是，删除G基因的36nt也会损害基因组复制。我们认为，G基因独特的结构特征为阐明调控核心蛋白与P蛋白翻译的调控机制提供了机会之窗。这项R21拨款申请的目标1将验证这一假设，即插入在核心基因Aug下游创建发夹结构以增强翻译起始。目的2将验证核心基因上游的一个小的开放阅读框架(UORF)作为P蛋白翻译的正调控因子。目的3将调查为什么G基因在前核心区有两个无义突变。除PG RNA外，HBV5‘端还可产生约30个核苷酸延伸的3.5kb RNA。这种前C区(PC)RNA专门翻译前C区/核心蛋白，它是乙肝e抗原(HBeAg)的前体。慢性乙肝晚期常选择前核心区G1896A无义突变来消除HBeAg的表达。奇怪的是，G型在密码子2处含有额外的C1817T无义突变。我们认为C1817T能够在uORF而不是核心基因重新启动翻译，从而将PC RNA重定向到P蛋白翻译，并挽救G型复制，尽管插入了36个核苷酸。我们的研究将阐明核心和P蛋白表达的翻译控制，并有助于理解不同的乙肝病毒基因变异如何避免偏离有效基因组复制所需的最佳核心/P蛋白比例。

DESCRIPTION (provided by applicant): Hepatitis B virus (HBV), which causes liver cirrhosis and hepatocellular carcinoma, has a small, compact genome. The only transcript required for genome replication is the 3.5-kb pregenomic (pg) RNA. It serves as the dicistronic mRNA for the translation of both core and P proteins, and also as the genome precursor to be encapsidated and converted by the P protein to double stranded DNA. Since a capsid is assembled from 240 copies of core protein but packages probably just one molecule of P protein, efficient genome replication requires a proper ratio of core / P protein translation. P protein translation involves ribosomal leaky scanning of upstream AUG codons including those of the core gene, but the control mechanisms remain ill defined. Genotype G harbors a unique 36-nt insertion at the 5' end of its core gene, which markedly enhances core protein translation whether in genotype G or when artificially introduced to other genotypes. This increase in core protein translation is rather associated with impaired genome replication in non-G genotypes, most likely due to a corresponding reduction in P protein translation. Paradoxically, deleting the 36nt from genotype G also impaired genome replication. We propose that the unique structural features of genotype G provide a window of opportunity to elucidate the control mechanisms regulating the translation of core vs. P protein. Aim 1 of this R21 grant application will verify the hypothesis that the insertion creates a hairpin structure downstream of core gene AUG to augment translation initiation. Aim 2 will validate a small open reading frame upstream of the core gene (the uORF) as a positive regulator of P protein translation. Aim 3 will investigate why genotype G harbors two nonsense mutations in the precore region. Besides pg RNA, HBV produces another 3.5-kb RNA with about 30-nt extension at the 5' end. This precore (pc) RNA is devoted exclusively to the translation of precore/core protein, the precursor to hepatitis B e antigen (HBeAg). The late stage of chronic HBV infection often selects for the G1896A nonsense mutation in the precore region to abolish HBeAg expression. Curiously, genotype G harbors an extra C1817T nonsense mutation at codon 2. We propose that C1817T enables translational re-initiation at the uORF instead of the core gene, thus redirecting the pc RNA towards P protein translation and rescuing genotype G replication despite the 36-nt insertion. Our studies will clarify translational control of core and P protein expression, and help understand how different HBV genetic variants avoid deviating from an optimal core/P protein ratio required for efficient genome replication.