The role of acylation of Gp5/M of porcine reproductive and respiratory syndrome virus for assembly and budding of virus particles elucidated by super resolution microscopy.

超分辨率显微镜阐明猪繁殖与呼吸综合征病毒 Gp5/M 酰化对病毒颗粒组装和出芽的作用。

• 批准号: 525482931
• 负责人: Dr. Susann Kummer
• 金额: --
• 依托单位: Institut für Virologie (WE05)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/525482931?language=en
• 关键词: role; acylation; Gp5; porcine; reproductive

Porcine respiratory and reproductive syndrome virus (PRRSV) is the most important pathogen in swine herds, but its membrane proteins and essential aspects of virus replication, such as virus budding, are poorly studied. In a previous DFG-project, we showed that the most abundant membrane protein of PRRSV, the Gp5/M dimer, is acylated at cysteines located at the end of the transmembrane region of Gp5 and M. The modification is essential for virus replication, especially for the assembly and release of virus particles. The enzymes that acylate Gp5/M have not been identified yet, but transfer of fatty acids to proteins is mediated by one or several of the 23 members of the DHHC family of proteins. We also used the artificial intelligence-based system of alphafold2 to predict a reliable structure of Gp5/M. It revealed six curved and tilted transmembrane helices, three from each protein. The third transmembrane helix extends into the cytoplasm and contains the acylation sites, which are located on the hydrophobic site of an amphiphilic helix. This resembles the amphiphilic helix of M2 of Influenza A virus, that inserts into the membrane to induce curvature. In this project, we aim to test the hypothesis that fatty acids bound to Gp5 and M are required for the clustering of Gp5/M dimers. We will use super-resolution microscopy to analyse whether Gp5/M dimers form oligomers at Golgi membranes and whether clustering is reduced when fatty acid attachment sites are removed from Gp5 and/or M. To achieve this goal, Susan Kummer, a specialist for STED microscopy from the Robert Koch Institute is a co-applicant. Mechanistically, the fatty acids might recruit cholesterol to the viral budding site, as has been recently described for acylation of the spike of SARS-CoV-2. This could produce a nanodomain in the membrane that promotes the assembly of further viral components, as has been described for the assembly of influenza viruses at the plasma membrane. This hypothesis will be tested with a photoactivated cholesterol analogue, which we have already used to identify a cholesterol binding site in the HA of influenza A viruses. We will also investigate the role of the amphiphilic helices of Gp5 and M for virus replication and acylation. Finally, we aim to identify the DHHC enzymes that acylate Gp5 and M. We hypothesise that DHHC 1, 4, 6 and/or 20, the only ER resident DHHCs, are the most likely candidates as Gp5 and M are acylated when expressed alone and not transported beyond the ER. We will use the same methods (siRNA and CRISPR/Cas9-mediated knock-out of DHHC genes) that we recently established to identify the DHHCs required for acylation of HA of Influenza A virus. This project not only provides molecular information for an important step in the replication cycle of Arteriviruses, but also clarifies similarities and differences in virus budding at internal membranes and at the plasma membrane.

猪呼吸与繁殖综合征病毒（Porcine respiratory and reproductive syndrome virus，PRRSV）是猪群中最重要的病原体，但其膜蛋白和病毒复制的重要方面，如病毒出芽，研究很少。在以前的DFG项目中，我们发现PRRSV最丰富的膜蛋白Gp 5/M二聚体在位于Gp 5和M跨膜区末端的半胱氨酸处被酰化。这种修饰对于病毒复制，特别是对于病毒颗粒的组装和释放是必不可少的。酰化Gp 5/M的酶尚未鉴定，但脂肪酸向蛋白质的转移由DHHC蛋白质家族的23个成员中的一个或几个介导。我们还使用了基于人工智能的alphafold 2系统来预测Gp 5/M的可靠结构。它揭示了六个弯曲和倾斜的跨膜螺旋，每个蛋白质三个。第三个跨膜螺旋延伸到细胞质中，并含有酰化位点，其位于两亲性螺旋的疏水位点上。这类似于甲型流感病毒M2的两亲性螺旋，插入膜中以诱导弯曲。在这个项目中，我们的目标是测试的假设，结合到Gp 5和M的脂肪酸所需的Gp 5/M二聚体的聚类。我们将使用超分辨率显微镜来分析Gp 5/M二聚体是否在高尔基体膜上形成寡聚体，以及当从Gp 5和/或M上去除脂肪酸附着位点时，聚簇是否减少。为了实现这一目标，罗伯特科赫研究所的STED显微镜专家Susan库默是共同申请人。从机制上讲，脂肪酸可能会将胆固醇招募到病毒出芽位点，正如最近描述的SARS-CoV-2尖峰的酰化。这可以在膜中产生纳米结构域，其促进其他病毒组分的组装，如已经描述的流感病毒在质膜上的组装。这一假设将与光活化的胆固醇类似物，我们已经用来确定在甲型流感病毒HA的胆固醇结合位点进行测试。我们还将研究Gp 5和M的两亲性螺旋对病毒复制和酰化的作用。最后，我们的目标是确定酰化Gp 5和M的DHHC酶。我们假设DHHC 1、4、6和/或20（仅有的ER驻留DHHC）是最可能的候选者，因为Gp 5和M在单独表达时被酰化并且不被转运到ER之外。我们将使用我们最近建立的相同方法（siRNA和CRISPR/Cas9介导的DHHC基因敲除）来鉴定甲型流感病毒HA酰化所需的DHHC。该项目不仅为动脉炎病毒复制周期中的重要步骤提供了分子信息，而且还澄清了病毒在内膜和质膜上出芽的相似性和差异。