Regulation of Virus Capsid Assembly

乙肝病毒是一种带有单链RNA中间体的包膜双链DNA病毒。正确组装 乙肝病毒的二十面体核心是复制所必需的。RNA前基因组的逆转录 (PgRNA)发生在完成的衣壳内，即核心的蛋白质外壳。乙肝病毒衣壳是 由120个衣壳蛋白(CP)二聚体组成。在体外，CP会自发组装。在体内，pgRNA是 在大量宿主RNA存在的情况下选择性地包裹，即使当CP在 互换。很难想象怎么会有选择性，特别是考虑到RNAbinding上的17个电荷 CP的结构域。是什么防止了错误组装？基于我们的体外研究，我们开发了 乙肝病毒装配受变构调控的假说：我们认为CP具有装配活性和- 不活跃的构象。我们已经确定了有利于或抑制组装的不同盐类和小分子。 我们还发现了一些抑制和增强组装的突变。同时，还有很高的 与产量的意外变化相关的CP的自然发生的突变的频率 病毒粒子；我们假设这些突变会影响组装的熟练程度。为了更好地了解 关于乙肝病毒组装的调节，我们建议检验我们的假设：(I)体外组装可以相关 与体内组装和(Ii)组装受变构调节。自然而然地聚集在一起 发生的突变将与表型相关。对于有装配缺陷的CP，我们将描述结构 Cp二聚体和Cp结合在衣壳中的差异，是变构的基本预测。我们会 研究二聚体界面的突变如何影响组装；通常，亚基之间的界面是 因变构转变而改变的。我们将研究CP的RNA结合和特异性。我们的初步数据 因为变构很强，但还需要进一步研究。我们对乙肝病毒的研究结果将推动 组装如何在其他病毒中受到调控。 超过3.5亿人患有慢性乙肝病毒(乙肝)感染，其中包括 超过125万美国人。在全球范围内，今年乙肝病毒将导致100万人死亡。通过确定 如何调控乙肝病毒组装，我们将确定抗病毒干预的新靶点。

Hepatitis B virus (HBV) is an enveloped dsDNA virus with a ssRNA intermediate form. Correct assembly of the icosahedral core of HBV is required for replication. Reverse transcription of the RNA pre-genome (pgRNA) takes place within the completed capsid, the protein shell of the core. The HBV capsid is constructed from 120 capsid protein (Cp) dimers. In vitro, Cp assembles spontaneously. In vivo, pgRNA is selectively encapsidated in the presence of a great excess of host RNA, even when the Cp is expressed in trans. It is hard to imagine how there can be any selectivity, especially given the +17 charge on the RNAbinding domain of Cp. What prevents misassembly? Based on our in vitro studies, we have developed the hypothesis that HBV assembly is allosterically regulated: we suggest that Cp has assembly-active and - inactive conformations. We have identified different salts and small molecules that favor or inhibit assembly. We have also identified a number of mutations that inhibit and enhance assembly. In parallel, there are high frequency, naturally occurring mutations of the Cp that correlate with unexpected changes in the yield of virions; we hypothesize that these mutations affect assembly proficiency. In order to better understand regulation of HBV assembly, we propose to test our hypotheses that (i) in vitro assembly can be correlated with assembly in vivo and (ii) assembly is subject to allosteric regulation. Assembly of the common naturally occurring mutants will be correlated with phenotype. With assembly-defective Cp, we will describe structural differences between Cp dimers and Cp bound in capsids, a fundamental prediction of allostery. We will investigate how mutations at the dimer interface affect assembly; typically, interfaces between subunits are altered by allosteric transitions. We will examine the RNA-binding and specificity of Cp. Our preliminary data for allostery is strong but requires further investigation. Our results with HBV will advance the question of how assembly is regulated in other viruses. More than 350 million individuals suffer from chronic infection with hepatitis B virus (HBV), including more than 1.25 million Americans. Worldwide, HBV will contribute to 1 million deaths this year. By determining how HBV assembly is regulated, we will characterize a new target for antiviral intervention.