Trafficking of Sendai virus nucleocapsids is mediated by intracellular vesicles.
Trafficking of Sendai virus nucleocapsids is mediated by intracellular vesicles.
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DOI：
10.1371/journal.pone.0010994
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发表时间：
2010-06-07
期刊：
影响因子：
3.7
通讯作者：
Takimoto T
中科院分区：
文献类型：
--
作者：
Chambers R;Takimoto T
副粘病毒是在细胞质中所有结构成分合成后，在质膜萌发部位组装而成。虽然病毒核糖核蛋白(VRNP)是感染性病毒粒子的重要组成部分，但vRNP转运到组装部位的过程尚不清楚。为了分析vRNPs在活体感染细胞中的实时转运，我们构建了重组仙台病毒rSeVLeGFP，该病毒融合了L蛋白和增强型绿色荧光蛋白(EGFP)。RSeVLeGFP的生长动力学与wt SeV相似，新合成的LeGFP最早可在感染后8h检测到。大多数LeGFP与vRNPs、NP和P蛋白的其他组分共定位，提示LeGFP的荧光信号代表vRNPs的位置。利用延时数字视频显微镜对LeGFP运动的分析揭示了LeGFP沿微管的方向性和跳跃性运动。诺康唑处理细胞限制了vRNP的运动，减少了子代病毒粒子的产生，但不影响病毒蛋白的合成，提示微管在vRNP运输和病毒组装中发挥作用。进一步的电子显微镜研究表明，vRNPs与感染细胞中存在的细胞内小泡密切相关。此外，vRNPs与Rab11a蛋白共定位，Rab11a蛋白调节循环内吞途径和高尔基体到质膜的转运。在感染的细胞中也观察到LeGFP和Rab11a之间的同步运动，这些细胞结构性地表达MRFP标记的Rab11a。VRNP与Alexa 594标记的循环转铁蛋白一起移动的事实也表明循环内体参与了vRNP的转位。综上所述，我们的结果强烈表明vRNP的细胞内囊泡运输途径参与了先前未知的转位，并为病毒结构成分到被膜病毒的组装部位的运输提供了新的见解。
Paramyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood. To analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594. Collectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses.
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