鸭源新城疫病毒M蛋白核定位信号突变影响病毒的毒力和复制能力

【目的】研究鸭源新城疫病毒(Newcastle disease virus,NDV)M蛋白核定位信号(nuclear localization signal,NLS)突变对其毒力和复制能力的影响。【方法】利用鸭源NDV SS1株P基因和F基因上的AgeⅠ和Bstz17Ⅰ酶切位点,将overlapPCR方法获得的M蛋白NLS突变的片段替换到p NDV/SS1GFP中获得全长质粒pNDV/SS1GFP-M/NLSm。通过反向遗传学技术拯救M蛋白NLS突变体病毒,并对拯救的病毒进行血凝(hemagglutination,HA)试验、荧光试验和M基因测序鉴定。另外,对突变体病毒进行M蛋白的亚细胞定位观察,以及病毒的生物学特性、空斑形成能力和体外增殖能力测定。【结果】成功构建M蛋白NLS突变的全长质粒pNDV/SS1GFP-M/NLSm。细胞转染物接种鸡胚后的第1代尿囊液无HA效价,盲传3代才能检测到拯救病毒的HA效价。进一步的荧光试验和M基因测序确定拯救的病毒是突变体病毒r SS1GFP-M/NLSm。与亲本病毒rSS1GFP相比,突变体病毒M蛋白由细胞核定位变为细胞质定位。此外,突变体病毒的毒力、在鸡胚上的复制能力以及在细胞中的空斑形成能力显著降低,并且感染细胞后产生的细胞病变轻微,M蛋白和绿色荧光蛋白的表达量均降低,说明M蛋白NLS突变使病毒的体外增殖能力受到抑制。【结论】NLS突变导致的M蛋白细胞核定位功能丧失可明显降低鸭源NDV的毒力和复制能力。

Nuclear localization signal mutation in the M protein attenuates the virulence and replication of duck-origin Newcastle disease virus

Objective] This study aimed to investigate the effect of nuclear localization signal (NLS) mutation in the M protein on the virulence and replication of duck-origin Newcastle disease virus (NDV). Methods] The target fragment owning M/NLS mutation was obtained by overlap PCR method and then used to replace the corresponding region of pNDV/SS1GFP to construct pNDV/SS1GFP-M/NLSm. The M/NLS mutant virus rSS1GFP-M/NLSm was rescued by reverse genetics technology and identified by hemagglutination (HA) assay, fluorescence assay and sequencing analysis of the M gene. The subcellular localization of M protein, biological characteristics, plaque formation ability and proliferation ability of the rescued virus were also detected. Results] The full-length plasmid pNDV/SS1GFP-M/NLSm was successfully constructed. The allantoic fluid obtained from the first generation had no HA titer, but the HA titer of the rescued virus was detected after three extra chicken egg passages. The results of fluorescence assay and M gene sequencing demonstrated that the rescued virus was rSS1GFP-M/NLSm. In comparison to the parental virus rSS1GFP, the M protein of rSS1GFP-M/NLSm mainly localized in the cytoplasm. In addition, the virulence, the replication ability and the plaque formation capacity of rSS1GFP-M/NLSm were obviously reduced. Moreover, the cytopathic effect and the expression of M protein and green fluorescent protein in rSS1GFP-M/NLSm-infected cells were also markedly decreased, indicating that the in vitro proliferation ability of duck-origin NDV was inhibited by M/NLS mutation. Conclusion] The disruption of M''s nuclear localization caused by M/NLS mutation could obviously attenuate the virulence and replication of duck-origin NDV.