One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis
One influenza virus particle packages eight unique viral RNAs as shown by FISH analysis
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DOI：
10.1073/pnas.1206069109
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发表时间：
2012-06-05
影响因子：
11.1
通讯作者：
Palese, Peter
中科院分区：
文献类型：
--
作者：
Chou, Yi-ying;Vafabakhsh, Reza;Palese, Peter
甲型流感病毒具有八个负义单链RNA的分段基因组。已显示这八个片段在病毒群体中以大致相等的摩尔比表示;然而，尚未确定每个病毒颗粒中每个病毒RNA片段的确切拷贝数。我们已经建立了一个实验方法的基础上，单分子荧光原位杂交（FISH）研究病毒RNA的组成在单病毒颗粒分辨率。共定位分析表明，高百分比的病毒颗粒包装所有八个不同的病毒RNA片段。为了确定单个病毒颗粒内每个RNA片段的拷贝数，我们测量了与靶向特定病毒RNA的荧光探针杂交的单个病毒颗粒的光漂白步骤。通过比较针对野生型流感A/波多黎各/8/34（PR 8）和携带两个拷贝的HA片段的重组PR 8病毒的HA RNA片段的探针的光漂白概况，我们得出结论，只有一个拷贝的HA片段被包装到野生型病毒颗粒中。我们的研究结果表明，其他RNA片段也有类似的光漂白行为，这表明对于大多数病毒颗粒来说，每个RNA片段只有一个拷贝被包装到一个病毒颗粒中。总之，我们的结果支持流感病毒基因组的包装是一个选择性过程。
Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.