Identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain VR-2332 for replication in cell culture
Identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain VR-2332 for replication in cell culture
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DOI：
10.1128/jvi.00562-07
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发表时间：
2007-09-01
影响因子：
5.4
通讯作者：
Faaberg, Kay S.
中科院分区：
文献类型：
--
作者：
Han, Jun;Liu, Gongping;Faaberg, Kay S.
猪繁殖与呼吸综合征病毒(PRRSV)的非结构蛋白2(NSP2)是一种多结构域蛋白，在其中间区域发生了显著的遗传变异，同时在N端推测的蛋白酶域和C端预测的跨膜区具有高度的保守性。建立了PRRSV北美原型VR-2332的反向遗传学系统，以探讨NSP2不同区域对病毒复制的重要性。设计了一系列nsp2编码区框架内缺失的突变体，随后从转基因细胞中回收了传染性病毒并对其进行了进一步鉴定。结果表明，半胱氨酸蛋白酶结构域(PL2)、PL2下游侧翼序列(氨基酸序列[AAL 181~323])和可能的跨膜结构域是复制的关键。相反，nsp2结构域之前的片段(aa13-35)对于病毒复制是不必要的，nsp2中间高变区(aa324-813)可以容忍100-aa或200-aa的缺失，但不能作为一个整体删除；最大的缺失约为400aa(nsp2 Delta 324-726)。对突变体的鉴定表明，小缺失的突变体具有与亲本病毒相似的生长动力学和核糖核酸表达谱，而NSP2 Delta 324726突变体对NL4、RC-145细胞的杀伤活性降低，没有形成可见的空斑。最后，通过将编码绿色荧光蛋白(GFP)的基因插入到一个NSP2缺失的突变体中，研究了NSP2的遗传灵活性在表达外源基因方面的利用。重组病毒是活的，但受损且不稳定，并通过大部分GFP基因的丢失逐渐获得亲本的生长动力学。
The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is a multidomain protein and has been shown to undergo remarkable genetic variation, primarily in its middle region, while exhibiting high conservation in the N-terminal putative protease domain and the C-terminal predicted transmembrane region. A reverse genetics system of PRRSV North American prototype VR-2332 was developed to explore the importance of different regions of nsp2 for viral replication. A series of mutants with in-frame deletions in the nsp2 coding region were engineered, and infectious viruses were subsequently recovered from transfected cells and further characterized. The results demonstrated that the cysteine protease domain (PL2), the PL2 downstream flanking sequence (amino acids [aal 181 to 323), and the putative transmembrane domain were critical for replication. In contrast, the segment of nsp2 preceding the PL2 domain (aa 13 to 35) was dispensable for viral replication, and the nsp2 middle hypervariable region (aa 324 to 813) tolerated 100-aa or 200-aa deletions but could not be removed as a whole; the largest deletion was about 400 aa (nsp2 Delta 324-726). Characterization of the mutants demonstrated that those with small deletions possessed growth kinetics and RNA expression profiles similar to those of the parental virus, while the nsp2 Delta 324726 mutant displayed decreased cytolytic activity on NL4,RC-145 cells and did not develop visible plaques. Finally, the utilization of the genetic flexibility of nsp2 to express foreign genes was examined by inserting the gene encoding green fluorescent protein (GFP) in frame into one nsp2 deletion mutant construct. The recombinant virus was viable but impaired and unstable and gradually gained parental growth kinetics by the loss of most of the GFP gene.