DynamicMembrane - Understanding mechanisms of membrane rupture and repair

在真核细胞内，膜隔室是封闭的实体，通过囊泡运输进行通信，由特定的蛋白质机制控制。作为专性细胞内寄生物，病毒在模拟囊泡形成的过程中从宿主获得其膜。核质大DNA病毒（NCLDV）是一个大DNA病毒家族，是这一规则的例外，并遵循非常规的膜获取途径。在感染过程中，它们形成开放的膜中间体，可能是膜破裂的结果，它们由此构建开放的膜球体。后者由病毒支架蛋白形成，这是大多数NCLDV中保守的蛋白质。膜球保持开放，直到病毒基因组被吸收，膜关闭，颗粒成熟为感染性病毒体。我们提出，这种不寻常的膜组装是由一种机制控制的，这种机制与NCLDV相同，包括（病毒）蛋白质和脂质。它们一起介导膜破裂、开放膜末端的稳定、开放球体的形成以及随后在DNA摄取后的闭合。 我们最近的脂质质谱（MS）的纯化的牛痘病毒（VACV，NCLDVs的成员）的结果表明，其包膜富含脂质物质，涉及膜破裂/不稳定。此外，通过3D电子断层扫描（ET）获得的初步数据确定了VACV蛋白，这是感染细胞膜破裂所需的。在这个建议的框架内，我们将寻找这种VACV蛋白在感染过程中的相互作用伙伴。 VACV蛋白将在有或没有其相互作用伴侣的情况下表达和纯化，并通过X射线晶体学以及冷冻EM分析其结构。对于前者，我们将依靠与Coulibaly博士（莫纳什，墨尔本）的合作，他是病毒蛋白，特别是VACV蛋白的X射线晶体学专家。同时，我们将继续脂质分析，并确定纯化VACV的次要脂质种类和特定脂质类别。我们建议通过脂质-MS的开放VACV膜，从感染的细胞中分离和亲和纯化，以补充这些数据。将在体外和体内分析特定脂质在膜破裂中的作用。在体外，破裂所需的VACV蛋白将在含有脂质的人工膜（脂质体）中重构，通过MS鉴定脂质将富集在病毒膜中。 采用含量释放法和冷冻-ET法分析其对脂质体的影响。 在体内，我们的目的是抑制感染细胞中特定脂质的合成，并评估ET对VACV组装的影响。我们的研究揭示了膜破裂和修复的机制，这些机制可能在细胞中靶向递送化合物中找到应用。 从进化的角度来看，它可能揭示了一种机制，这种机制要么在现代真核生物中罕见，要么存在于早期真核生物/古细菌/细菌中，但在进化过程中丢失了。

Within eukaryotic cells membrane compartments are closed entities that communicate by vesicular transport, controlled by specific protein machineries. Being obligatory intracellular parasites, viruses acquire their membrane from the host, in a process mimicking the formation of vesicles. Nucleo-cytoplasmic large DNA viruses (NCLDVs), a family of large DNA viruses, are an exception to this rule and follow an unconventional pathway of membrane acquisition. During infection they form open membrane intermediates, likely the result of membrane rupture, from which they build an open membrane sphere. The latter is shaped by the viral scaffold protein, a protein conserved among most NCLDVs. The membrane sphere remains open until the viral genome has been taken up, upon which the membrane closes and the particle matures into an infectious virion. We proposed that this unusual membrane assembly is controlled by a machinery, common to NCLDVs, which includes both (viral) proteins and lipids. Together they mediate membrane rupture, stabilization of open membrane ends, the formation of the open sphere and its subsequent closure after DNA-uptake. Our recent lipid mass spectrometry (MS) results of purified vaccinia virus (VACV, member of the NCLDVs) showed that its envelope is enriched in lipid species that have been implicated in membrane rupture/destabilization. Moreover, preliminary data obtained by 3D-electron tomography (ET) identified a VACV protein, required for membrane rupture in infected cells. Within the frame of this proposal we will search for interacting partners of this VACV protein during infection. The VACV-protein will be expressed and purified with or without its interacting partners and its structure analyzed by X-ray crystallography as well as by cryo-EM. For the former we will rely on the collaboration with Dr. Coulibaly (Monash, Melbourne), an expert on X-ray crystallography of viral proteins, in particular of VACV proteins. In parallel we will continue lipid-analyses and identify minor lipid species and specific lipid classes of purified VACV. We propose to complement these data by lipid-MS of open VACV membranes, isolated and affinity-purified from infected cells. The role of specific lipids in membrane rupture will be analyzed in vitro as well as in vivo. In vitro the VACV protein required for rupture will be reconstituted in artificial membranes (liposomes) containing lipids identify by MS to be enriched in the viral membrane. Its effect on liposomes will be analyzed by content release and cryo-ET. In vivo we aim at inhibiting the synthesis of specific lipids in infected cells and assess effects on VACV assembly by ET. Our study sheds light on mechanisms of membrane rupture and repair that may find applications in targeted delivery of compounds in cells. Evolutionary, it may shed light on a mechanism that is either rare in modern eukaryotes or was present in an early eukaryote/archaea/bacteria but was lost during evolution.