Lu Y E, Cassese T, Kielian M
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Virol. 1999 May;73(5):4272-8. doi: 10.1128/JVI.73.5.4272-4278.1999.
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 approximately 100-fold in infection and approximately 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.
塞姆利基森林病毒（SFV）和辛德毕斯病毒（SIN）是包膜α病毒，它们通过内吞途径中低pH触发的融合进入细胞，并通过从质膜出芽而离开。先前对胆固醇耗尽的昆虫细胞的研究表明，SFV在病毒融合和子代病毒的有效释放过程中都需要细胞膜中的胆固醇。一种SFV突变体srf-3，由于E1刺突亚基中的单个点突变（脯氨酸226突变为丝氨酸），在没有胆固醇的情况下仍能高效融合和释放。我们在此研究了胆固醇在SIN（一种与SFV关系较远的α病毒）的进入和释放过程中的作用。与对照细胞相比，胆固醇耗尽的细胞中SIN的生长、初次感染、融合和释放均受到显著抑制。基于SFV、srf-3和SIN之间E1 226区域内的序列差异，我们构建了该区域内发生改变的六个SIN突变体，并对它们的胆固醇依赖性进行了表征。一个SIN突变体SGM，其从E1位置224到235具有srf-3氨基酸序列，与野生型SIN的感染和融合相比，在胆固醇耗尽的细胞中的感染增加了约100倍，融合增加了约250倍。脉冲追踪分析表明，SGM从胆固醇耗尽的细胞中的释放明显比野生型SIN更有效。因此，与SFV类似，SIN在病毒进入和释放过程中都依赖胆固醇，并且这两个步骤的胆固醇依赖性都可以通过E1 226区域内的序列进行调节。
