Hepatitis B virus transcriptional interference and liver cancer-related mutations

 描述（由申请方提供）：慢性B型肝炎病毒（HBV）感染的建立需要通过B e抗原（HBeAg）和过量包膜蛋白（以亚病毒颗粒的形式）诱导免疫耐受。随后的免疫清除阶段选择核心启动子突变和SPII启动子缺失。这两种类型的突变和HBx蛋白，一种转录反式激活因子，已被牵连在肝细胞癌（HCC）的发展。核心启动子驱动HBeAg的3.5-kb pc RNA和基因组复制的稍短的pg RNA的转录，而SPII启动子驱动中等和小（S）包膜蛋白的2.1-kb RNA。核心启动子突变减少HBeAg表达，但通过以pc RNA为代价上调pg RNA的转录来增强基因组复制。令人惊讶的是，赋予基因组复制最高水平的突变降低了S蛋白水平。由于所有四种类型的HBV转录物都是单向的，并且在3'末端是共末端的，我们的解释是3.5-kb RNA通过启动子封闭干扰2.1-kb RNA的转录。为了验证这一假设，将确定删除核心启动子或将其替换为强CMV启动子对2.1-kb RNA转录的影响。为了检测2.1 kb RNA是否干扰0.7 kb RNA对HBx蛋白的转录，我们将确定在HCC患者中发现的SPII启动子缺失是否上调HBx转录。最后，由于所有转录物的3'末端与末端冗余的3.5-kb RNA的5'末端重叠，所以丰富的2.1-kb RNA具有抑制3.5-kb RNA转录的潜力。这将通过检查与载体连接的二聚体构建体相比，在环化HBV基因组的背景下SPII启动子缺失的复制影响来验证。我们建议，双向转录干扰允许HBV有效地协调其基因组复制与包膜和HBx蛋白表达。我们以前发现核心启动子突变使HBx蛋白单独表达（并以高水平表达）能够触发细胞增殖。 转录干扰将允许SPII启动子缺失显著增加来自完整基因组的HBx蛋白表达。因此，这两种类型的突变可能与HBx蛋白协同作用，促进肝癌发生。总之，本申请探索了HBV转录调控的新机制，同时将HCC发展中的三种病毒因子联系起来。

 DESCRIPTION (provided by applicant): Establishment of chronic hepatitis B virus (HBV) infection requires induction of immune tolerance by hepatitis B e antigen (HBeAg) and excess envelope proteins in the form of subviral particles. The subsequent immune clearance phase rather selects for core promoter mutations and SPII promoter deletions. Both types of mutations and HBx protein, a transcriptional transactivator, have been implicated in the development of hepatocellular carcinoma (HCC). The core promoter drives transcription of the 3.5-kb pc RNA for HBeAg and slightly shorter pg RNA for genome replication, while the SPII promoter drives the 2.1-kb RNA for the middle and small (S) envelope proteins. Core promoter mutations diminish HBeAg expression but enhance genome replication through transcriptional up regulation of pg RNA at the expense of pc RNA. Surprisingly, mutations conferring the highest level of genome replication reduced S protein level. Since all the four classes of HBV transcripts are unidirectional and co-terminal at the 3' end, our interpretation is that the 3.5-kb RNAs interfere with transcription of the 2.1-kb RNA by promoter occlusion. To verify this hypothesis, the impact of deleting the core promoter or replacing it with the strong CMV promoter on transcription of the 2.1-kb RNA will be determined. To examine whether the 2.1-kb RNA interferes with transcription of the 0.7-kb RNA for HBx protein, we will establish whether SPII promoter deletions found in HCC patients up regulate HBx transcript. Finally, since the 3' end of all the transcripts overlaps with the 5' end of the terminally redundant 3.5-kb RNAs, the abundant 2.1-kb RNA has the potential to inhibit transcription of the 3.5-kb RNAs. This will be verified by examining the replication impact of SPII promoter deletions in the context of a circularized HBV genome in contrast to vector-linked dimeric construct. We propose that bidirectional transcriptional interference allows HBV to effectively coordinate its genome replication with envelope and HBx protein expression. We previously found that core promoter mutations enable the HBx protein expressed alone (and at high level) to trigger cell proliferation. Transcriptional interference will allow SPII promoter deletions to markedly augment HBx protein expression from the intact genome. Thus, the two types of mutations could work synergistically with HBx protein to promote hepatocarcinogenesis. In summary, this application explores a novel mechanism of HBV transcriptional regulation and at the same time connects the three viral factors in HCC development.