Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells
Biarsenical Labeling of Vesicular Stomatitis Virus Encoding Tetracysteine-Tagged M Protein Allows Dynamic Imaging of M Protein and Virus Uncoating in Infected Cells
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DOI：
10.1128/jvi.01668-08
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发表时间：
2009-03-15
影响因子：
5.4
通讯作者：
Pattnaik, Asit K.
中科院分区：
文献类型：
--
作者：
Das, Subash C.;Panda, Debasis;Pattnaik, Asit K.
重组水疱性口炎病毒（VSV-PeGFP-M-MmRFP）编码增强的与P融合在框架中的绿色荧光蛋白（PeGFP）代替P，在G-L基因连接处编码融合基质蛋白（单体红色荧光蛋白融合在M的羧基端[MmRFP]），以及正常位置的野生型（wt） M蛋白，但MmRFP未掺入病毒粒子中。随后，我们生成了用羧基端四胱氨酸标签（Mtc）代替M蛋白编码M蛋白的重组病毒（VSV-PeGFP-Delta M-Mtc和VSV-Delta M-Mtc）。这些重组病毒以与wt VSV中的M相似的水平结合Mtc，表明传染性横纹肌病毒编码并结合标记的M蛋白的恢复。被VSV-PeGFP-Delta M-Mtc感染的细胞释放出用双砷红染料（ReAsH）标记的病毒粒子呈双荧光，荧光绿色是由于PeGFP在核衣壳中结合，而荧光红色是由于ReAsH标记的Mtc在病毒包膜中结合。M蛋白的转运和随后与质膜的结合被证明是独立于微管的。用双砷染料ReAsH和FlAsH（绿色）对感染VSV-Delta M- mtc的细胞进行序列标记，发现新合成的M蛋白在不到30分钟的时间内到达质膜，并在质膜上持续积累长达2个半小时。使用双荧光VSV，我们确定在质膜上吸附后，一半的病毒颗粒进入细胞并在细胞质中剥离其核衣壳所需的时间约为28分钟。
A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-Delta M-Mtc and VSV-Delta M-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-Delta M-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-Delta M-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.