Bayer N, Schober D, Prchla E, Murphy R F, Blaas D, Fuchs R
Department of General and Experimental Pathology, University of Vienna, Vienna, Austria.
J Virol. 1998 Dec;72(12):9645-55. doi: 10.1128/JVI.72.12.9645-9655.1998.
Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.
巴弗洛霉素A1（baf）是液泡质子ATP酶的特异性抑制剂，通常用于证明内体低pH值对病毒脱壳的必要性。然而，在某些细胞类型中，baf也会影响内吞物质从早期内吞区室到晚期内吞区室的转运。为了表征常用于研究病毒感染早期事件的HeLa细胞中的内吞途径，我们使用了35S标记的人鼻病毒2型（HRV2）以及各种液相标记物。这些病毒粒子通过受体介导的内吞作用被摄取，并在pH值<5.6时发生构象变化成为C抗原颗粒，导致基因组RNA释放并最终引发感染（E. Prchla、E. Kuechler、D. Blaas和R. Fuchs，《病毒学杂志》68:3713 - 3723，1994）。荧光显微镜和自由流电泳（FFE）对微粒体进行亚细胞分级分离结果显示，baf使所有标记物在早期内体中的转运停滞。相反，发现破坏微管的药物诺考达唑通过使标记物在内体载体囊泡（ECV）中积累来抑制转运，ECV是早期和晚期内体之间的一个区室。因此，HRV2的溶酶体降解受到抑制，而其构象变化和感染性不受该药物影响。通过FFE分析在诺考达唑存在下HRV2和液相标记物的亚细胞分布，发现与不存在诺考达唑的对照孵育没有差异。因此，ECV和晚期内体具有相同的电泳迁移率，腔内pH值<5.6且允许HRV2脱壳。由于巴弗洛霉素不仅会消除内体的低pH值，还会阻断HeLa细胞中从早期内体到晚期内体的转运，其对病毒感染的抑制作用部分也可能归因于病毒被困在早期内体中，而早期内体可能缺乏脱壳所需的成分。因此，不能仅将巴弗洛霉素对病毒脱壳的抑制作用视为表明需要低pH值。
