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.

猪呼吸与生殖综合征病毒(PRRSV)是猪群中最重要的病原体，但对其膜蛋白和病毒萌发等病毒复制的关键环节的研究较少。在之前的DFG项目中，我们发现PRRSV最丰富的膜蛋白Gp5/M二聚体是在Gp5和M跨膜区末端的半胱氨酸上被酰化的，这种修饰对于病毒复制，特别是病毒颗粒的组装和释放是必不可少的。使Gp5/M酰化的酶尚未确定，但脂肪酸向蛋白质的转移是由DHHC家族23个成员中的一个或几个成员介导的。我们还使用基于人工智能的Alphafold2系统来预测Gp5/M的可靠结构。它显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，含有酰化位点，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，插入膜中诱导弯曲。在这个项目中，我们的目标是检验这一假设，即Gp5和M结合的脂肪酸是Gp5/M二聚体聚集所必需的。我们将使用超分辨率显微镜来分析Gp5/M二聚体是否在高尔基膜上形成低聚物，以及当脂肪酸从Gp5和/或M中移除时，聚集是否会减少。为了实现这一目标，罗伯特·科赫研究所的STED显微镜专家Susan Kummer是共同申请者之一。从机理上讲，脂肪酸可能会将胆固醇募集到病毒的萌芽部位，就像最近被描述的对SARS-CoV-2尖峰的酰化一样。这可能会在膜上产生一个纳米结构域，促进更多病毒成分的组装，就像流感病毒在质膜上组装所描述的那样。这一假说将用一种光激活的胆固醇类似物来检验，我们已经用它来确定甲型流感病毒HA中的胆固醇结合部位。我们还将研究Gp5和M的两亲性螺旋对病毒复制和酰化的作用。最后，我们的目标是鉴定使Gp5和M酰化的DHHC酶。我们假设，只有内质网驻留的DHHC 1、4、6和/或20是最有可能的候选，因为Gp5和M在单独表达时是酰化的，而不是运输到内质网之外。我们将使用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。该项目不仅为动脉病毒复制周期中的一个重要步骤提供了分子信息，而且还阐明了病毒在内膜和质膜上发芽的异同。

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.