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

描述（由申请人提供）：乙型肝炎病毒（HBV）会导致肝硬化和肝细胞癌，其基因组小而紧凑。基因组复制所需的唯一转录物是 3.5 kb 前基因组 (pg) RNA。它作为核心蛋白和 P 蛋白翻译的双顺反子 mRNA，也是被 P 蛋白衣壳化并转化为双链 DNA 的基因组前体。由于衣壳是由 240 个核心蛋白拷贝组装而成，但可能只包装一个 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 对上游 AUG 密码子（包括核心基因的密码子）进行核糖体渗漏扫描，但控制机制仍不明确。基因型 G 在其核心基因的 5' 端有一个独特的 36-nt 插入，无论是在基因型 G 中还是在人工引入其他基因型时，都显着增强了核心蛋白的翻译。核心蛋白翻译的增加与非 G 基因型中基因组复制受损相当相关，很可能是由于 P 蛋白翻译的相应减少所致。矛盾的是，从基因型 G 中删除 36nt 也会损害基因组复制。我们认为，G 基因型的独特结构特征为阐明调节核心蛋白与 P 蛋白翻译的控制机制提供了一个机会之窗。该 R21 资助申请的目标 1 将验证以下假设：插入在核心基因 AUG 下游产生发夹结构以增强翻译起始。目标 2 将验证核心基因上游的一个小型开放阅读框 (uORF) 作为 P 蛋白翻译的正调节因子。目标 3 将研究为什么基因型 G 在前核心区域存在两个无义突变。除了 pg RNA 之外，HBV 还产生另一种 3.5 kb RNA，在 5' 端延伸约 30 nt。该前核心 (pc) RNA 专门用于翻译前核心/核心蛋白，即乙型肝炎 e 抗原 (HBeAg) 的前体。慢性HBV感染的晚期常常选择前核心区的G1896A无义突变来消除HBeAg表达。奇怪的是，基因型 G 在密码子 2 处有一个额外的 C1817T 无义突变。我们提出，C1817T 能够在 uORF 而不是核心基因上重新启动翻译，从而将 pc RNA 重定向到 P 蛋白翻译并挽救基因型 G 的复制，尽管插入了 36 个核苷酸。我们的研究将阐明核心蛋白和 P 蛋白表达的翻译控制，并帮助了解不同的 HBV 遗传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.