Institut für Virologie, Freie Universität Berlin, Berlin, Germany.
College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.
PLoS Pathog. 2021 Apr 23;17(4):e1009554. doi: 10.1371/journal.ppat.1009554. eCollection 2021 Apr.
Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped positive-strand RNA virus in the Arteiviridae family, is a major pathogen affecting pigs worldwide. The membrane (glyco)proteins GP5 and M form a disulfide-linked dimer, which is a major component of virions. GP5/M are required for virus budding, which occurs at membranes of the exocytic pathway. Both GP5 and M feature a short ectodomain, three transmembrane regions, and a long cytoplasmic tail, which contains three and two conserved cysteines, respectively, in close proximity to the transmembrane span. We report here that GP5 and M of PRRSV-1 and -2 strains are palmitoylated at the cysteines, regardless of whether the proteins are expressed individually or in PRRSV-infected cells. To completely prevent S-acylation, all cysteines in GP5 and M have to be exchanged. If individual cysteines in GP5 or M were substituted, palmitoylation was reduced, and some cysteines proved more important for efficient palmitoylation than others. Neither infectious virus nor genome-containing particles could be rescued if all three cysteines present in GP5 or both present in M were replaced in a PRRSV-2 strain, indicating that acylation is essential for virus growth. Viruses lacking one or two acylation sites in M or GP5 could be rescued but grew to significantly lower titers. GP5 and M lacking acylation sites form dimers and GP5 acquires Endo-H resistant carbohydrates in the Golgi apparatus suggesting that trafficking of the membrane proteins to budding sites is not disturbed. Likewise, GP5 lacking two acylation sites is efficiently incorporated into virus particles and these viruses exhibit no reduction in cell entry. We speculate that multiple fatty acids attached to GP5 and M in the endoplasmic reticulum are required for clustering of GP5/M dimers at Golgi membranes and constitute an essential prerequisite for virus assembly.
猪繁殖与呼吸综合征病毒（PRRSV）是动脉炎病毒科的有包膜正链 RNA 病毒，是一种影响全球猪群的主要病原体。膜（糖）蛋白 GP5 和 M 形成二硫键连接的二聚体，是病毒粒子的主要成分。GP5/M 是病毒出芽所必需的，出芽发生在细胞外途径的膜上。GP5 和 M 的特征均为短的胞外结构域、三个跨膜区和一个长的细胞质尾巴，其细胞质尾巴中分别有三个和两个保守的半胱氨酸，靠近跨膜区。我们在此报告，PRRSV-1 和 -2 株的 GP5 和 M 均在半胱氨酸上发生棕榈酰化，无论这些蛋白是单独表达还是在 PRRSV 感染的细胞中表达。要完全阻止 S-酰化，GP5 和 M 中的所有半胱氨酸都必须交换。如果 GP5 或 M 中的单个半胱氨酸被取代，则棕榈酰化减少，并且一些半胱氨酸比其他半胱氨酸对有效棕榈酰化更为重要。如果在 PRRSV-2 株中替换 GP5 或 M 中存在的所有三个或两个半胱氨酸，则既不能拯救感染性病毒也不能拯救含有基因组的颗粒，表明酰化对于病毒生长是必需的。缺失 M 或 GP5 中一个或两个酰化位点的病毒可以被拯救，但生长到明显较低的滴度。缺乏酰化位点的 GP5 和 M 形成二聚体，并且 GP5 在高尔基体内获得内酰胺酶抗性碳水化合物，表明膜蛋白向出芽部位的运输不受干扰。同样，缺乏两个酰化位点的 GP5 被有效地掺入病毒粒子中，并且这些病毒在细胞进入中没有减少。我们推测，内质网中附着在 GP5 和 M 上的多个脂肪酸对于 GP5/M 二聚体在高尔基膜上的聚集是必需的，并且是病毒组装的必要前提。
