Means R E, Desrosiers R C
Department of Microbiology and Molecular Genetics, New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102, USA.
J Virol. 2000 Dec;74(23):11181-90. doi: 10.1128/jvi.74.23.11181-11190.2000.
Stocks of simian immunodeficiency virus (SIV) from the supernatants of infected cell cultures were used to examine the sensitivity of envelope glycoprotein gp120 to enzymatic deglycosylation and the effects of enzyme treatment on infectivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis revealed little or no change in the mobility of virion-associated gp120 after digestion with high concentrations of N-glycosidase F, endoglycosidase F, endoglycosidase H, and endo-beta-galactosidase. Soluble gp120, which was not pelletable after the enzymatic reaction, was sensitive to digestion by the same enzymes within the same reaction mix and was only slightly less sensitive than gp120 that had been completely denatured by boiling in the presence of SDS and beta-mercaptoethanol. Digestion by three of the seven glycosidases tested significantly changed the infectivity titer compared to that of mock-treated virus. Digestion by endo-beta-galactosidase increased infectivity titers by about 2.5-fold, and neuraminidase from Newcastle disease virus typically increased infectivity titers by 8-fold. Most or all of the increase in infectivity titer resulting from treatment with neuraminidase could be accounted for by effects on the virus, not the cells; SIV produced in the presence of the sialic acid analog 2,3-dehydro-2-deoxy-N-acetylneuraminic acid also exhibited increased infectivity, and the effects could not be duplicated by neuraminidase treatment of cells. Digestion with mannosidase reduced infectivity by fivefold. Our results indicate that carbohydrates on native oligomeric gp120 as it exists on the surface of virus particles are largely occluded and are refractory to digestion by glycosidases. Furthermore, the sialic acid residues at the ends of carbohydrate side chains significantly reduce the inherent infectivity of SIV.
来自受感染细胞培养上清液的猿猴免疫缺陷病毒（SIV）毒株被用于检测包膜糖蛋白gp120对酶促去糖基化的敏感性以及酶处理对感染性的影响。十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳和蛋白质印迹分析显示，用高浓度的N-糖苷酶F、内切糖苷酶F、内切糖苷酶H和内切β-半乳糖苷酶消化后，病毒体相关gp120的迁移率几乎没有变化或没有变化。酶促反应后不可沉淀的可溶性gp120在相同反应混合物中对相同酶的消化敏感，并且仅比在SDS和β-巯基乙醇存在下煮沸而完全变性的gp120稍不敏感。与模拟处理的病毒相比，所测试的七种糖苷酶中的三种酶的消化显著改变了感染性滴度。用内切β-半乳糖苷酶消化使感染性滴度提高了约2.5倍，新城疫病毒的神经氨酸酶通常使感染性滴度提高8倍。用神经氨酸酶处理导致的感染性滴度的大部分或全部增加可能是由于对病毒而非细胞的影响；在唾液酸类似物2,3-脱氢-2-脱氧-N-乙酰神经氨酸存在下产生的SIV也表现出感染性增加，并且这种影响不能通过对细胞进行神经氨酸酶处理来复制。用甘露糖苷酶消化使感染性降低了五倍。我们的结果表明，病毒颗粒表面天然寡聚体gp120上的碳水化合物在很大程度上被封闭，并且对糖苷酶的消化具有抗性。此外，碳水化合物侧链末端的唾液酸残基显著降低了SIV的固有感染性。
