目前，我们已经清楚知道HIV感染机体细胞的策略，运用显微镜的技术，我们可以轻松获取病毒的三维立体结构，对于我们研究病毒感染有很大的帮助。因为我们受限于可见光的波长，因此，运用传统的光学显微镜我们并不能观察到小于200nm的病毒结构，病毒的结构一般为25至300nm。
用荧光标记的靶蛋白，这样在特定的时间激活荧光，然后可以将所有荧光标记的位置以一种复合图像显现出来，荧光标记物可以干扰所标记蛋白的功能，这样，使得我们研究活性蛋白的功能变得困难起来。
近日，巴黎巴斯的研究所的研究者Nathalie Arhel和他的同事们使用一种改良的技术将六个氨基酸残基的结构单元插入到了HIV使用的一种酶中，HIV使用这种酶来将自己的DNA整合入宿主的基因组中，这种6个氨基酸的结构单元因为非常短，可以影响这种酶的功能，如果足够长的话可以结合到荧光标记分子上。
这种新的技术可以帮助我们更近一步的研究HIV，帮助我们更深入地和HIV进行对话，以前，我们并不清楚HIV病毒是如何将自己的遗传物质释放到宿主的细胞质中的，然而研究者当前的技术可以帮助我们理解这一过程，这种技术可以在病毒DNA整合至宿主基因组之前对病毒进行定位，给我们提供更多的研究信息。相关研究成果刊登在了近日的国际杂志PNAS上。（生物谷：T.Shen编译）
doi:10.1073/pnas.1013267109
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Superresolution imaging of HIV in infected cells with FlAsH-PALM
Mickaël Leleka,1, Francesca Di Nunziob,1, Ricardo Henriquesa, Pierre Charneaub, Nathalie Arhelb,2, and Christophe Zimmera,2
Imaging protein assemblies at molecular resolution without affecting biological function is a long-standing goal. The diffraction-limited resolution of conventional light microscopy (∼200–300 nm) has been overcome by recent superresolution (SR) methods including techniques based on accurate localization of molecules exhibiting stochastic fluorescence; however, SR methods still suffer important restrictions inherent to the protein labeling strategies. Antibody labels are encumbered by variable specificity, limited commercial availability and affinity, and are mostly restricted to fixed cells. Fluorescent protein fusions, though compatible with live cell imaging, substantially increase protein size and can interfere with their biological activity. We demonstrate SR imaging of proteins tagged with small tetracysteine motifs and the fluorescein arsenical helix binder (FlAsH-PALM). We applied FlAsH-PALM to image the integrase enzyme (IN) of HIV in fixed and living cells under experimental conditions that fully preserved HIV infectivity. The obtained resolution (∼30 nm) allowed us to characterize the distribution of IN within virions and intracellular complexes and to distinguish different HIV structural populations based on their morphology. We could thus discriminate ∼100 nm long mature conical cores from immature Gag shells and observe that in infected cells cytoplasmic (but not nuclear) IN complexes display a morphology similar to the conical capsid. Together with the presence of capsid proteins, our data suggest that cytoplasmic IN is largely present in intact capsids and that these can be found deep within the cytoplasm. FlAsH-PALM opens the door to in vivo SR studies of microbial complexes within host cells and may help achieve truly molecular resolution.
