State Key Laboratory of Pathogen and Biosecurity, Institute of Epidemiology and Microbiology, Academy of Millitary Medical Sciences, Beijing, China.
PLoS One. 2013 Apr 8;8(4):e61166. doi: 10.1371/journal.pone.0061166. Print 2013.
Coronaviruses are a family of large positive-sense RNA viruses that are responsible for a wide range of important veterinary and human diseases. Nsp1 has been shown to have an important role in the pathogenetic mechanisms of coronaviruses in vivo. To assess the function of a relatively conserved domain (LLRKxGxKG) of MHV nsp1, a mutant virus, MHV-nsp1-27D, with a 27 nts (LLRKxGxKG) deletion in nsp1, was constructed using a reverse genetic system with a vaccinia virus vector. The mutant virus had similar growth kinetics to MHV-A59 wild-type virus in 17CI-1 cells, but was highly attenuated in vivo. Moreover, the mutant virus completely protected C57BL/6 mice from a lethal MHV-A59 challenge. To further analyze the mechanism of the attenuation of the mutant virus, changes in reporter gene expression were measured in nsp1- or nsp1-27D-expressing cells; the results showed that nsp1 inhibited reporter gene expression controlled by different promoters, but that this inhibition was reduced for nsp1-27D. The research in vivo and in vitro suggests that the LLRKxGxKG region of nsp1 may play an important role in this process.
冠状病毒是一类大型正链 RNA 病毒，可导致广泛的重要兽医和人类疾病。研究表明，Nsp1 在冠状病毒的体内致病机制中具有重要作用。为了评估 MHV nsp1 中相对保守结构域（LLRKxGxKG）的功能，我们使用带有痘苗病毒载体的反向遗传学系统构建了一个缺失 nsp1 中 27 个核苷酸（LLRKxGxKG）的突变病毒 MHV-nsp1-27D。在 17CI-1 细胞中，该突变病毒的生长动力学与 MHV-A59 野生型病毒相似，但在体内高度减毒。此外，该突变病毒可完全保护 C57BL/6 小鼠免受致死性 MHV-A59 攻击。为了进一步分析突变病毒减毒的机制，我们在表达 nsp1 或 nsp1-27D 的细胞中测量了报告基因表达的变化；结果表明，nsp1 抑制了由不同启动子控制的报告基因表达，但这种抑制作用在 nsp1-27D 中降低。体内和体外的研究表明，nsp1 的 LLRKxGxKG 区域可能在这一过程中发挥重要作用。
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