Replication and secretion of hepatitis B virus variants

描述（由申请人提供）：乙型肝炎病毒（HBV）感染全球4亿人，并导致肝炎，肝硬化和肝癌。HBV相关的肝脏疾病是病毒与其宿主复杂相互作用的结果。核心启动子突变体是普遍存在的HBeAg表达降低的HBV变体，在感染的HBe晚期和抗HBe阶段取代了野生型菌株。这种突变体与暴发性肝炎有关。核心启动子突变体的复制能力仍然存在争议。目前的方法将最常见的1762/1764核心启动子突变引入野生型HBV基因组，因此在遗漏基因组中其他核心启动子突变和共同进化突变方面存在缺陷。我们的方法的新颖之处在于测试自然发生的核心启动子突变体是否比野生型菌株更有效地复制。我们已经确定了两个核心启动子突变体（4B和3.4），其复制水平分别比野生型分离物高10倍和5倍。两者都携带1762/1764和其他突变。4B分泌病毒颗粒到培养上清液的效率是3.4的4倍。4B同时分泌包膜病毒颗粒和裸核颗粒，而3.4不能分泌包膜颗粒。目前的研究计划进一步表征自然发生的核心启动子突变的复制和分泌表型。首先，我们将研究自然发生的核心启动子突变体的复制能力。我们计划确定高复制能力是否是自然发生的核心启动子突变体的共同特征。负责增强4B复制的序列将通过与低复制克隆的嵌合结构缩小。在另一种单独的方法中，核心启动子突变对高复制表型的贡献将被直接验证。其次，将确定3.4和4B不同分泌表型的机制。负责的序列元素将通过3.4和4B之间的马赛克构造进行映射。至于分泌的改变是由变异的表面蛋白还是核心蛋白介导的，还有待确定。这里研究的病毒复制和分泌是病毒的基本特征，对疾病的严重程度、治疗反应和恢复有深远的影响。复制的增加和分泌的减少可能有助于诱发包括暴发性肝炎在内的严重肝脏疾病。

DESCRIPTION (provided by applicant): Hepatitis B virus (HBV) infects 400 million people worldwide and causes hepatitis, cirrhosis, and liver cancer. HBV related liver diseases are the outcome of complex interplay between the virus and its host. Core promoter mutants are prevalent HBV variants with reduced HBeAg expression, which replace the wildtype strains in the late HBe and anti-HBe stages of infection. Such mutants have been implicated in fulminant hepatitis. The replication capacity of core promoter mutants remains controversial. The current approach introduces the most common core promoter mutations at 1762/1764 into wild-type HBV genome, and is thus flawed in omitting other core promoter mutations and co-evolved mutations elsewhere in the genome. The novelty of our approach is to test whether naturally occurring core promoter mutants replicate more efficiently than wild-type strains. We have identified two core promoter mutants (4B and 3.4) that replicated at 10 and 5 fold higher levels than wild-type isolates, respectively. Both harbored 1762/1764 and additional mutation(s). 4B secreted viral particles to culture supernatant at 4 times higher efficiency than 3.4. Moreover, 4B secreted both enveloped viral particles and naked core particles while 3.4 failed to secrete enveloped particles. The present study plans to further characterize the replication and secretion phenotypes of naturally occurring core promoter mutants. First, we will study the replication capacity of naturally occurring core promoter mutants. We plan to establish whether high replication capacity is a common feature of naturally occurring core promoter mutants. The sequence responsible for enhanced replication of 4B will be narrowed down by chimeric constructs with a low replicating clone. In a separate approach, the contribution of core promoter mutations to the high replication phenotype will be verified directly. Second, the mechanism for different secretion phenotypes of 3.4 versus 4B will be determined. The responsible sequence element will be mapped by mosaic constructs between 3.4 and 4B. Whether altered secretion is mediated by variant surface or core protein will be determined. The viral replication and secretion as studied here are basic features of the virus with profound effect on disease severity, response to therapy and recovery. Increase in replication and decrease in secretion may help induce severe liver diseases including fulminant hepatitis.