Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus
Intracellular synthesis, processing, and transport of proteins encoded by ORFs 5 to 7 of porcine reproductive and respiratory syndrome virus
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DOI：
10.1006/viro.1996.0356
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发表时间：
1996-07-01
期刊：
影响因子：
3.7
通讯作者：
Dea, S
中科院分区：
文献类型：
--
作者：
Mardassi, H;Massie, B;Dea, S
猪繁殖与呼吸综合征病毒（PRRSV）是一种含有正链RNA基因组的小包膜病毒，具有至少三种主要结构蛋白N、M和5。N蛋白被认为是核衣壳的主要成分，而M和E是与膜相关的。先前的研究使用肽特异性抗体将这些蛋白分别定位于orf 7、6和5。本研究利用单特异性抗血清对大肠杆菌表达的orf5、orf6和orf7产物进行培养，在高度允许的MARC-145细胞系中研究了PRRSV结构蛋白的合成和加工。用多种糖苷酶处理病毒蛋白表明，只有E被n链聚糖修饰。脉冲追踪实验显示，主要包膜糖蛋白的细胞内转运在内侧前高尔基区室被延迟。在前30分钟的追踪过程中，5的表观分子量逐渐向下移动，这被认为是由于富含甘露糖的聚糖结构被修剪的结果。一旦E被运输到内侧高尔基体或近端元件，一些分子将其所有高甘露糖n -连接的低聚糖完全加工成复合物类型，而在其他分子中，只有一小部分n -连接的聚糖最终糖基化。这两种不同糖基化形式的E被发现与细胞外病毒粒子结合。在细胞和病毒粒子中，M和5都出现在由二硫键连接的异二聚体复合物中。从脉冲追踪实验中分析的寡聚化过程表明，M和E以不同的动力学和效率结合到M-E复合物中，其方式类似于马动脉炎病毒中的对应物。显然，5种蛋白n -聚糖加工的所有步骤都是在其与内质网（ER）中的M结合后进行的。在感染细胞中，E和M表现为与膜高度相关，而N主要是细胞质。(C) 1996学术出版社，Inc.
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a small enveloped virus containing a positive-strand RNA genome, possesses at least three major structural proteins designated N, M, and 5. The N protein is considered as the major component of the nucleocapsid, whereas M and E are membrane-associated. Previous studies using peptide-specific antibodies assigned these proteins to ORFs 7, 6, and 5, respectively. In the present report, monospecific antisera raised against Escherichia coli-expressed ORFs 5, 6, and 7 products were used to study the synthesis and processing of PRRSV structural proteins in the highly permissive MARC-145 cell line, Treatment of viral proteins with various glycosidases showed that only E was modified by N-linked glycans. Pulse-chase experiments revealed that intracellular transport of the major envelope glycoprotein was delayed in the premedial Golgi compartmenl. During the first 30 min of chase, 5 undergoes a gradual downward shift of its apparent molecular weight, thought to result from trimming of the mannose-rich glycan structures, Once E is transported to the medial Golgi or proximal elements, some molecules undergo complete processing of all their high-mannose N-linked oligosaccharides to complex type, while in other molecules only a fraction of N-linked glycans are terminally glycosylated. These two differentially glycosylated forms of E were found to be incorporated into extracellular virions. In cells and virions, both M and 5 were shown to occur in heterodimeric complexes linked by disulfide bonds, The oligomerization process, as analyzed from pulse-chase experiments, showed that M and E are incorporated into M-E complexes with different kinetics and efficiencies, in a fashion similar to their counterparts in equine arteritis virus. Apparently, all steps of 5 protein N-glycans processing proceed after its association with M which occurs in the endoplasmic reticulum (ER). In the infected cells, E and M appear highly membrane-associated, while N is predominantly cytosolic. (C) 1996 Academic Press, Inc.