Liposomal Nanocontainers as Models for Viral Infection: Monitoring Viral Genomic RNA Transfer through Lipid Membranes
Liposomal Nanocontainers as Models for Viral Infection: Monitoring Viral Genomic RNA Transfer through Lipid Membranes
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DOI：
10.1128/jvi.00329-11
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发表时间：
2011-08-01
影响因子：
5.4
通讯作者：
Blaas, Dieter
中科院分区：
文献类型：
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作者：
Bilek, Gerhard;Matscheko, Nena M.;Blaas, Dieter
摄取到靶细胞后，许多无包膜病毒在内吞室的低 pH 环境中经历构象变化。这导致两亲性病毒肽和/或疏水蛋白结构域的暴露，这些肽和/或疏水蛋白结构域插入并破坏或穿孔囊泡膜。病毒核酸由此进入细胞质并开始复制。我们在这里演示了将人鼻病毒2型单链正义RNA基因组体外转移到用重组极低密度脂蛋白受体片段修饰的脂质体中。膜附着的病毒粒子暴露于 pH 5.4，模拟晚期内涵体的体内 pH 环境。这触发了 RNA 的释放，RNA 到达脂质体腔时可通过封装的逆转录酶通过原位 cDNA 合成进行检测。随后，对cDNA进行PCR扩增。当病毒粒子与脂质的比例较低时，RNA 转移与病毒浓度呈正相关。然而，在病毒浓度较高时，膜会出现渗漏，从而导致 cDNA 合成减少。根据早期的体内数据，在生理相关的病毒浓度下，RNA 穿过脂质膜，不会对囊泡造成严重损害。
After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.