Replication and secretion of hepatitis B virus variants

乙型肝炎病毒变异体的复制和分泌

• 批准号: 6557207
• 负责人: SHUPING TONG
• 金额: $ 15.4万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6557207
• 关键词: chimeric proteins; density gradient ultracentrifugation; gene mutation; genetic mapping; genetic promoter element; genetic strain; hepatitis B; hepatitis B virus group; host organism interaction; microorganism culture; molecular cloning; phenotype; polymerase chain reaction; radioimmunoassay; secretion; site directed mutagenesis; southern blotting; transfection; virulence; virus infection mechanism; virus protein; virus replication

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.

描述（由申请人提供）：乙型肝炎病毒 (HBV) 感染全球 4 亿人，导致肝炎、肝硬化和肝癌。乙型肝炎相关的肝脏疾病是病毒与其宿主之间复杂相互作用的结果。核心启动子突变体是流行的 HBV 变异体，其 HBeAg 表达降低，在感染晚期和抗 HBe 阶段取代野生型菌株。此类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1762/1764处最常见的核心启动子突变引入野生型HBV基因组中，因此在忽略基因组其他地方的其他核心启动子突变和共同进化突变方面存在缺陷。我们方法的新颖之处在于测试天然存在的核心启动子突变体是否比野生型菌株复制更有效。我们已经鉴定出两个核心启动子突变体（4B 和 3.4），它们的复制水平分别比野生型分离株高 10 倍和 5 倍。两者均含有 1762/1764 和其他突变。 4B分泌病毒颗粒至培养上清液的效率比3.4高4倍。此外，4B同时分泌有包膜的病毒颗粒和裸露的核心颗粒，而3.4则不能分泌有包膜的颗粒。本研究计划进一步表征天然存在的核心启动子突变体的复制和分泌表型。首先，我们将研究天然存在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天然存在的核心启动子突变体的共同特征。负责增强 4B 复制的序列将通过具有低复制克隆的嵌合构建体来缩小范围。在另一种方法中，将直接验证核心启动子突变对高复制表型的贡献。其次，将确定3.4与4B的不同分泌表型的机制。负责的序列元素将通过 3.4 和 4B 之间的镶嵌构建体进行映射。将确定改变的分泌是否由变异表面或核心蛋白介导。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、治疗反应和恢复具有深远影响。复制的增加和分泌的减少可能有助于诱发严重的肝脏疾病，包括暴发性肝炎。