Viswanathan Kasinath, Verweij Marieke C, John Nessy, Malouli Daniel, Früh Klaus
Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, United States of America.
PLoS One. 2017 Nov 9;12(11):e0187899. doi: 10.1371/journal.pone.0187899. eCollection 2017.
Human cytomegalovirus (HCMV) depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC), membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM) cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells.
人巨细胞病毒（HCMV）在病毒生命周期的每个阶段都依赖并调节多种宿主细胞膜蛋白。为了全面了解HCMV感染对膜蛋白的影响，我们使用氨基酸稳定同位素标记（SILAC）、膜分离以及二维液相色谱和串联质谱法进行蛋白质鉴定，分析了HCMV感染引起的成纤维细胞膜蛋白丰度变化。这种系统方法揭示，感染期间CD81、CD44、CD98、小窝蛋白-1和连环蛋白δ-1下调，而GRP-78上调。由于在用紫外线灭活病毒感染期间也观察到CD81下调，我们推测这种四跨膜蛋白是病毒进入过程的一部分。有趣的是，在HCMV进入期间，四跨膜蛋白家族的其他成员CD9和CD151也下调。由于富含四跨膜蛋白的微区（TEM）聚集宿主细胞膜蛋白，包括已知的CMV受体如整合素，我们研究了TEM是否是病毒进入所必需的。当用胆固醇螯合剂甲基-β-环糊精破坏TEM时，病毒进入受到抑制，这种抑制与CD81、CD9和CD151表面水平降低相关，而整合素水平保持不变。此外，同时用siRNA介导的多种四跨膜蛋白敲低抑制了病毒进入，而单独敲低几乎没有效果，这表明单个四跨膜蛋白在进入过程中起重要但冗余的作用。总之，我们的数据表明TEM作为HCMV进入细胞所利用的受体平台。
