The cholesterol requirement for Sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence
The cholesterol requirement for Sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence
复制标题
DOI：
10.1128/jvi.73.5.4272-4278.1999
复制
发表时间：
1999-05-01
影响因子：
5.4
通讯作者：
Kielian, M
中科院分区：
文献类型：
--
作者：
Lu, YPE;Cassese, T;Kielian, M
塞姆利基森林病毒(SFV)和辛德比斯病毒(SIN)都是被囊膜的甲型病毒，它们通过低pH触发的内吞途径融合进入细胞，然后通过出芽离开质膜。以前对胆固醇耗尽的昆虫细胞的研究表明，SFV需要细胞膜中的胆固醇来实现病毒融合和后代病毒的有效退出。SFV突变体SRF-3在没有胆固醇的情况下显示出有效的融合和退出，这是由于E1尖峰亚单位Pro 226到丝氨酸的单点突变。我们在这里描述了胆固醇在SIN进出中的作用，SIN是一种与SFV关系密切的甲型病毒，与对照细胞相比，胆固醇耗竭细胞中SIN的生长、原发感染、融合和退出都受到显著抑制。基于SFV、SRF-3和SIN在E1226区域的序列差异，我们构建了6个在该区域发生改变的SIN突变体，并对它们的胆固醇依赖性进行了表征。与野生型SIN的感染和融合相比，SRF-3氨基酸序列从224位到235位的SIN突变体SGM在感染和与胆固醇耗竭细胞融合方面显示出类似100倍的增加和250倍的融合。脉冲追逐分析表明，SGM从胆固醇耗竭细胞中退出的效率明显高于野生型SIN。因此，与SFV相似，SIN对病毒的进入和退出都是胆固醇依赖的，这两个步骤的胆固醇依赖都可以由E1226区域内的序列调节。
Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped alphaviruses that enter cells via low-pH-triggered fusion in the endocytic pathway and exit by budding from the plasma membrane. Previous studies with cholesterol-depleted insect cells have shown that SFV requires cholesterol in the cell membrane for both virus fusion and efficient exit of progeny virus. An SFV mutant, srf-3, shows efficient fusion and exit in the absence of cholesterol due to a single point mutation in the E1 spike subunit, proline 226 to serine. We have here characterized the role of cholesterol in the entry and exit of SIN, an alphavirus quite distantly related to SFV, Growth, primary infection, fusion, and exit of SIN were all dramatically inhibited in cholesterol-depleted cells compared to control cells. Based on sequence differences within the E1 226 region between SFV, srf-3, and SIN, we constructed six SIN mutants with alterations within this region and characterized their cholesterol dependence. A SIN mutant, SGM, that had the srf-3 amino acid sequence from E1 position 224 to 235 showed increases of similar to 100-fold in infection and similar to 250-fold in fusion with cholesterol-depleted cells compared with infection and fusion of wild-type SIN. Pulse-chase analysis demonstrated that SGM exit from cholesterol-depleted cells was markedly more efficient than that of wild-type SIN. Thus, similar to SFV, SIN was cholesterol dependent for both virus entry and exit, and the cholesterol dependence of both steps could be modulated by sequences within the E1 226 region.