A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity
A DNA-launched reverse genetics system for porcine reproductive and respiratory syndrome virus reveals that homodimerization of the nucleocapsid protein is essential for virus infectivity
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DOI：
10.1016/j.virol.2004.10.026
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发表时间：
2005-01-05
期刊：
影响因子：
3.7
通讯作者：
Yoo, D
中科院分区：
文献类型：
--
作者：
Lee, C;Calvert, JG;Yoo, D
反向遗传系统是针对高毒力“非典型”猪繁殖与呼吸综合征病毒（PRRSV）而开发的。 15 395 个核苷酸的全长基因组被组装为单个 cDNA 克隆，并置于原核 T7 或真核 CMV 启动子下。用 F NITA 转录物或 DNA 克隆转染细胞可诱导细胞病变效应并产生感染性后代。重组的病毒是稳定的并且在细胞中生长至亲本病毒的滴度。感染后，猪产生PRRSV典型的临床症状和肺部病理学，并诱导病毒血症和特异性抗体。之前，我们表明 PRRSV 核衣壳 (N) 蛋白通过非共价和共价相互作用形成同二聚体，并且第 23 位的半胱氨酸负责共价相互作用。使用感染性 cDNA 克隆评估 N 半胱氨酸对 PRRSV 感染性的功能意义。 N的第23、75和90位的每个半胱氨酸被丝氨酸取代，并且将单个突变掺入到cDNA克隆中，从而构建了三个独立的半胱氨酸突变体。转染时，野生型和 C75S 克隆诱导细胞病变效应并产生具有难以区分的噬斑形态的感染性病毒。相反，C23S 突变完全消除了克隆的感染性，表明 C23 介导的 N 蛋白同源化在 PRRSV 感染性中发挥着关键作用。出乎意料的是，C90S 突变似乎对病毒感染性也具有致命性。复制缺陷型 C23S 和 C90S 突变体的基因组复制和 mRNA 转录均呈阳性。数据表明。除了同二聚化之外，PRRSV N 蛋白还可以使用半胱氨酸 90 与另一种结构蛋白进行异二聚化，并且 N 蛋白异二聚化对于 PRRSV 感染性至关重要 (C) 2004 Elsevier Inc. 保留所有权利。
Reverse genetic systems were developed for a highly virulent 'atypical' porcine reproductive and respiratory syndrome virus (PRRSV). The full-length genome of 15 395 nucleotides was assembled as a single cDNA clone and placed under either the prokaryotic T7 or eukaryotic CMV promoter. Transfection of cells with the F NITA transcripts or the DNA clone induced cytopathic effects and produced infectious progeny. The reconstituted virus was stable and grew to the titer of the parental virus in cells. Upon infection, pigs produced clinical signs and lung pathology typical for PRRSV and induced viremia and specific antibodies. Previously, we showed that the PRRSV nucleocapsid (N) protein forms homodimers via both noncovalent and covalent interactions and that cysteine at position 23 is responsible for die covalent interaction. The functional significance of cysteines of N for PRRSV infectivity was assessed using the infectious cDNA clone. Each cysteine of N at positions 23, 75, and 90 was replaced with serine and the individual mutation was incorporated into the cDNA clone such that three independent cysteine mutants were constructed. When transfected, the wild type and C75S clones induced cytopathic effects and produced infectious virus with indistinguishable plaque morphology. In contrast, the C23S mutation completely abolished infectivity of the clone indicating that C23-mediated N protein homodinierization plays a critical role in PRRSV infectivity. Unexpectedly, the C90S mutation also appeared to be lethal for virus infectivity. Genome replication and mRNA transcription were both positive for the replication-defective C23S and C90S mutants. The data suggest that. in addition to homodimerization, the PRRSV N protein may also undergo heterodimerization with another structural protein using cysteine 90 and that the N protein heterodimerization is essential for PRRSV infectivity (C) 2004 Elsevier Inc. All rights reserved.