Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

逆转录病毒（Retrovirus）的组装由多结构域结构蛋白Gag驱动。Gag的衣壳结构域（capsid domains, CA）之间的相互作用可介导Gag多聚化，进而形成由基于二聚体、三聚体与六聚体蛋白接触的蛋白质晶格构成的未成熟病毒粒子。不同逆转录病毒之间的六聚体内外接触存在差异，尤其在CA的N端亚结构域（CANTD）中表现显著。针对HIV-1，细胞内分子肌醇六磷酸（inositol hexakisphosphate, IP6）可结合未成熟六聚体并使其稳定，且该分子是感染性病毒粒子产生所必需的。本研究借助体外组装、冷冻电子断层扫描（cryo-electron tomography）与亚断层平均（subtomogram averaging）技术、全原子分子动力学模拟（atomistic molecular dynamics simulations）以及突变分析，对与HIV相关的慢病毒——马传染性贫血病毒（equine infectious anemia virus, EIAV）展开研究。我们的核心目标在于解析未成熟慢病毒晶格的结构保守性，以及IP6在EIAV组装过程中的作用。与HIV-1类似，IP6可显著促进EIAV Gag蛋白在体外组装为病毒样颗粒（virus-like particles, VLPs），这些颗粒呈现三种形态差异显著的类型：窄管、宽管与球形。对上述VLPs进行亚4埃分辨率的结构表征后，我们意外发现三种形态均基于保留关键结构组件的未成熟晶格，这凸显了CA形成未成熟组装体的结构通用性。直接对比EIAV与HIV的结果显示，两种慢病毒均拥有相似的未成熟互作界面，该界面由保守与非保守残基共同介导构建。在EIAV与HIV-1中，IP6均通过CACTD与SP区域内的保守赖氨酸残基调控未成熟组装过程。最后，我们证实IP6可刺激慢病毒属中其他数种逆转录病毒的未成熟粒子体外组装，这表明IP6在慢病毒组装过程中存在保守的功能角色。