DynamicMembrane - Understanding mechanisms of membrane rupture and repair

在真核细胞中，膜室是封闭的实体，由特定的蛋白质机制控制，通过囊泡运输进行通信。作为必须的细胞内寄生虫，病毒从宿主那里获得膜，在一个模仿小泡形成的过程中。核质大DNA病毒(NCLDV)是一类大型DNA病毒，是这一规则的例外，它遵循一种非传统的膜获取途径。在感染期间，它们形成开放的膜中间体，很可能是膜破裂的结果，从那里它们建立一个开放的膜球体。后者是由病毒支架蛋白塑造的，这种蛋白在大多数NCLDV中都是保守的。膜球体保持开放，直到病毒基因组被占据，在此之后膜关闭，颗粒成熟为具有感染性的病毒粒子。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一样，包括(病毒)蛋白质和脂质。它们共同调节膜的破裂、开放膜末端的稳定、开放球的形成以及DNA摄取后的关闭。我们最近对纯化的痘苗病毒(VACV)的脂质谱(MS)结果表明，其包膜富含与膜破裂/不稳定有关的脂类物种。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必需的。在这项提案的框架内，我们将寻找这种VACV蛋白在感染过程中的相互作用伙伴。VACV-蛋白将在有或没有相互作用伙伴的情况下进行表达和纯化，并通过X射线结晶学和冷冻-EM分析其结构。对于前者，我们将依靠与Coulibaly博士(墨尔本莫纳什)的合作，他是病毒蛋白质，特别是VACV蛋白质的X射线结晶学专家。同时，我们将继续进行脂类分析，并鉴定纯化的VACV的少量脂类和特定的脂类。我们建议通过从感染细胞分离和亲和纯化的开放VACV膜的脂质谱来补充这些数据。特定的脂类在膜破裂中的作用将在体外和体内进行分析。在体外，破裂所需的VACV蛋白将在含有MS鉴定的富含在病毒膜中的脂类的人造膜(脂质体)中重组。其对脂质体的影响将通过内容释放和冷冻来分析。在体内，我们的目标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，这可能会在化合物在细胞中的靶向递送中得到应用。在进化上，它可能揭示一种机制，这种机制在现代真核生物中很罕见，或者在早期的真核生物/古生菌/细菌中存在，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.