Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture

Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.

泡沫病毒（Foamy Viruses, FV）隶属于泡沫病毒属（Spumavirus），在逆转录病毒科（Retroviridae）中构成一个独特的演化支系。尽管其自然宿主感染以及跨物种传播至人类的过程均呈无症状状态，但FV可在人类细胞中高效复制，使其成为极具潜力的基因治疗载体候选者。本研究报道了纯化的原型泡沫病毒（Prototype Foamy Virus, PFV）以及PFV感染细胞的冷冻电子显微镜（cryo-electron microscopy）与（冷冻）电子断层成像超微结构数据。成熟PFV颗粒具有独特形态：衣壳尺寸恒定，且在衣壳与包膜之间存在一层有序性较低的致密壳层，该结构大概率由Gag N端结构域以及Env前导肽gp18LP的胞质部分构成。病毒包膜中含有三聚体Env糖蛋白，这些糖蛋白部分排列为相互连接的六边形组装体。通过对野生型Env以及Env gp48TM-gp80SU切割位点突变体进行亚断层平均，我们得到了原位三维重建结构，结果显示二者的刺突结构相似，且相邻三聚体的低密度区域间存在清晰连接，从而稳定了六边形晶格。研究人员利用冷冻电镜技术获得了处于预融合状态的糖蛋白的9埃分辨率结构，该结构揭示了位于刺突顶部的受体结合亚基gp80SU所介导的广泛三聚体相互作用，以及源自融合蛋白亚基gp48TM的三根中心螺旋。Env的下部区域，推测由gp48TM、gp80SU与gp18LP的交错部分组成，将刺突锚定在包膜上。我们推测gp48TM的致密结构延伸为三根中心跨膜螺旋，后者与源自gp18LP的三根外侧跨膜螺旋相互作用。本研究的超微结构数据与9埃分辨率的糖蛋白结构，为阐明PFV的分子架构及其与逆转录病毒科其他成员间独特的演化关系提供了重要的新见解。