目前,，我們已經(jīng)清楚知道HIV感染機(jī)體細(xì)胞的策略，運(yùn)用顯微鏡的技術(shù),，我們可以輕松獲取病毒的三維立體結(jié)構(gòu),，對(duì)于我們研究病毒感染有很大的幫助。因?yàn)槲覀兪芟抻诳梢?jiàn)光的波長(zhǎng),，因此,，運(yùn)用傳統(tǒng)的光學(xué)顯微鏡我們并不能觀察到小于200nm的病毒結(jié)構(gòu)，病毒的結(jié)構(gòu)一般為25至300nm,。
用熒光標(biāo)記的靶蛋白,，這樣在特定的時(shí)間激活熒光，然后可以將所有熒光標(biāo)記的位置以一種復(fù)合圖像顯現(xiàn)出來(lái),，熒光標(biāo)記物可以干擾所標(biāo)記蛋白的功能,，這樣，使得我們研究活性蛋白的功能變得困難起來(lái),。
近日,，巴黎巴斯的研究所的研究者Nathalie Arhel和他的同事們使用一種改良的技術(shù)將六個(gè)氨基酸殘基的結(jié)構(gòu)單元插入到了HIV使用的一種酶中，HIV使用這種酶來(lái)將自己的DNA整合入宿主的基因組中,，這種6個(gè)氨基酸的結(jié)構(gòu)單元因?yàn)榉浅６?，可以影響這種酶的功能,，如果足夠長(zhǎng)的話可以結(jié)合到熒光標(biāo)記分子上。
這種新的技術(shù)可以幫助我們更近一步的研究HIV,，幫助我們更深入地和HIV進(jìn)行對(duì)話,，以前，我們并不清楚HIV病毒是如何將自己的遺傳物質(zhì)釋放到宿主的細(xì)胞質(zhì)中的,，然而研究者當(dāng)前的技術(shù)可以幫助我們理解這一過(guò)程,，這種技術(shù)可以在病毒DNA整合至宿主基因組之前對(duì)病毒進(jìn)行定位，給我們提供更多的研究信息,。相關(guān)研究成果刊登在了近日的國(guó)際雜志PNAS上,。（生物谷：T.Shen編譯）
doi:10.1073/pnas.1013267109
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PMID:
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.
