Identification of temperature-sensitive mutations in the phosphoprotein of respiratory syncytial virus that are likely involved in its interaction with the nucleoprotein
Identification of temperature-sensitive mutations in the phosphoprotein of respiratory syncytial virus that are likely involved in its interaction with the nucleoprotein
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DOI：
10.1128/jvi.76.6.2871-2880.2002
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发表时间：
2002-03-01
影响因子：
5.4
通讯作者：
Jin, H
中科院分区：
文献类型：
--
作者：
Lu, B;Brazas, R;Jin, H
人呼吸道合胞病毒（RSV）的磷蛋白（P）是病毒RNA聚合酶的重要组成部分，沿着的还有大聚合酶（L）、核衣壳（N）和M2-1蛋白。通过筛选随机诱变的P基因cDNA文库，在酵母双杂交试验中，鉴定了两个独立的突变，一个是172位甘氨酸被丝氨酸取代（G172 S），另一个是176位谷氨酸被甘氨酸取代（E176 G），这两个突变导致了37 ℃下N-P相互作用的丧失。两种P突变体在37和39 ℃下支持RSV微型基因组复制子的复制和转录的活性大大降低。将G172 S和E176 G突变分别引入RSV A2（rA 2）反基因组cDNA中，获得重组病毒rA 2-P172和rA 2-P176。这两种病毒在33 ℃下在Vero和HEp-2细胞中复制以及中间型A2病毒，但每种突变病毒在两种细胞系中均表现出温度敏感性复制。rA 2-P176比rA 2-P172对温度更敏感。从病毒感染的细胞的N蛋白与每个P突变体的免疫共沉淀表明，N-P相互作用在37 degreesC受损。此外，rA 2-P172和rA 2-P176在小鼠和棉鼠肺中的复制水平降低。与体外试验的情况一样，rA 2-P176在小鼠和棉鼠下呼吸道中的复制比rA 2-P172更受限制。在37 ℃的体外传代过程中，rA 2-P176中的E176 G突变迅速从甘氨酸变为主要的天冬氨酸;还检测到半胱氨酸或丝氨酸的突变。所有的回复突变体都失去了温度敏感的表型。为了分析从位置161至180的区域中的氨基酸对于P蛋白功能的重要性，引入额外的突变并在体外分析它们的功能。在微型基因组测定中，含有P基因中的G172 S和E176 G变化的双突变体、在位置174至176处的三个带电残基被丙氨酸取代以及从位置161至180处的残基缺失完全消除了P蛋白功能。因此，在位置172和176处的氨基酸和相邻的带电残基在P蛋白的功能中起关键作用。
The phosphoprotein (P) of human respiratory syncytial virus (RSV) is an essential component of the viral RNA polymerase, along with the large polymerase (L), nucleocapsid (N), and M2-1 proteins. By screening a randomly mutagenized P gene cDNA library, two independent mutations, one with a substitution of glycine at position 172 by serine (G172S) and the other with a substitution of glutamic acid at position 176 by glycine (E176G), were identified to result in the loss of N-P interaction at 37degreesC in the yeast two-hybrid assay. Both P mutants exhibited greatly reduced activity in supporting the replication and transcription of an RSV minigenome replicon at 37 and 39degreesC. The G172S and E176G mutations were introduced individually into the RSV A2 (rA2) antigenomic cDNA, and recombinant viruses, rA2-P172 and rA2-P176, were obtained. Both viruses replicate as well as Mid-type A2 virus in both Vero and HEp-2 cells at 33degreesC, but each mutant virus exhibited temperature-sensitive replication in both cell lines. rA2-P176 is more temperature sensitive than rA2-P172. Coimmunoprecipitation of the N protein with each P mutant from virus-infected cells demonstrates that N-P interaction is impaired at 37degreesC. In addition, the levels of replication of rA2-P172 and rA2-P176 in the lungs of mice and cotton rats were reduced. As is the case with the in vitro assays, rA2-P176 is more restricted in replication in the lower respiratory tract of mice and cotton rats than rA2-P172. During in vitro passage at 37degreesC, the E176G mutation In rA2-P176 was rapidly changed from glycine to predominantly aspartic acid; mutations to cysteine or serine were also detected. All of the revertants lost the temperature-sensitive phenotype. To analyze the importance of the amino acids in the region from positions 161 to 180 for the P protein function, additional mutations were introduced and their functions were analyzed in vitro. A double mutant containing both G172S and E176G changes in the P gene, substitution of the three charged residues at positions 174 to 176 by alanine, and a deletion of residues from positions 161 to 180 completely abolished the P protein function in the minigenome assay. Thus, the amino acids at positions 172 and 176 and the adjacent charged residues play critical roles in the function of the P protein.