Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells
Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells
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DOI：
10.1128/jvi.00211-06
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发表时间：
2006-07-01
影响因子：
5.4
通讯作者：
Pattnaik, Asit K.
中科院分区：
文献类型：
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作者：
Das, Subash C.;Nayak, Debasis;Pattnaik, Asit K.
水疱性口炎病毒（VSV）的磷酸化蛋白(P)是病毒RNA聚合酶的一个亚基。在之前的研究中，我们证明在蛋白的铰链区插入19个氨基酸对P蛋白的功能没有显著影响。在本研究中，我们将框架中的全长增强绿色荧光蛋白（eGFP）插入到P的铰链区，并表明融合蛋白（PeGFP）在病毒基因组转录和复制中具有功能，尽管活性降低。重组水疱性口炎病毒编码PeGFP代替P蛋白（VSV-PeGFP），与野生型VSV相比，其生长动力学降低。利用重组VSV-PeGFP，我们发现病毒复制蛋白和新合成的RNA共定位到整个细胞质的位置，表明复制和转录不局限于细胞质的任何特定区域。被egfp标记的病毒感染的细胞的实时成像显示，在合成后，核衣壳通过微管（MT）介导的过程向细胞周围运输，并且核衣壳被认为与线粒体密切相关。用nocodazole或Colcemid（已知可抑制MT聚合的药物）处理细胞，导致核衣壳在细胞核周围积聚，也导致传染性病毒的产生受到抑制。这些发现与一种模型相一致，在这种模型中，子代病毒核衣壳通过MT向细胞外周运输，这种运输可能由线粒体促进。
The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.