The baculovirus GP64 envelope fusion protein: synthesis, oligomerization, and processing.
The baculovirus GP64 envelope fusion protein: synthesis, oligomerization, and processing.
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DOI：
10.1006/viro.1995.1291
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发表时间：
1995-06
期刊：
影响因子：
3.7
通讯作者：
A. Oomens;S. Monsma;G. Blissard
中科院分区：
文献类型：
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作者：
A. Oomens;S. Monsma;G. Blissard
杆状病毒GP64包膜融合蛋白（GP64 EFP）是一类完整的膜蛋白，它进入分泌途径，在转运到质膜的过程中被寡聚化和广泛加工。采用脉冲标记、脉冲追踪和免疫沉淀实验等方法，研究了假杉树多衣壳核多角体病毒（OpMNPV）感染的Lymantria dispar细胞中GP64 EFP的生物合成、寡聚和加工动力学。在整个感染周期的不同时间，检测了感染opmnpv的L. dispar细胞中GP64 EFP的相对合成率。利用脉冲标记法和免疫沉淀法，检测到GP64 EFP在2小时内合成，并在24-26小时内观察到最大的合成速率，这一时间与感染opmnpv的L. dispar细胞开始高水平产生出芽病毒的时间一致。为了确定GP64 EFP的低聚结构，制备了一种可溶形式的OpMNPV GP64 EFP，并通过凝胶过滤色谱、非还原SDS-PAGE和质谱相结合的方法进行了检测。低聚物GP64 EFP被鉴定为三聚体分子，在非还原性SDS-PAGE上以两个离散带迁移。在感染周期的早期（12小时）和晚期（36小时）进行的脉冲追踪研究表明，GP64 EFP在合成后15分钟内完成寡聚化。然而，低聚效率相对较低，合成的GP64 EFP转化为三聚体的比例不到33%。大部分留在细胞内的GP64 EFP单体在合成后30 ~ 45分钟内降解。对感染后早期（12小时）和后期（36小时）碳水化合物加工动力学的分析表明，在感染的早期和晚期，碳水化合物都是快速添加的，并在GP64 EFP合成后10 ~ 20分钟开始加工。在早期阶段，碳水化合物加工在合成后大约90分钟内完成，而在后期阶段，同样的过程需要大约150分钟。因此，随着感染的进展，碳水化合物的处理效率似乎降低了。因此，这些研究表明GP64 EFP经历了一个快速但低效的寡聚步骤，导致GP64 EFP具有三聚体结构。虽然碳水化合物的添加是快速的，但碳水化合物的处理需要较长的时间（一半为45至75分钟），并且在感染后期似乎效率较低。
The baculovirus GP64 envelope fusion protein (GP64 EFP) is a class I integral membrane protein that enters the secretory pathway and is oligomerized and extensively processed during transport to the plasma membrane. The kinetics of GP64 EFP biosynthesis, oligomerization, and processing in Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV)-infected Lymantria dispar cells were examined by pulse label, pulse-chase, and immunoprecipitation experiments. Relative rates of GP64 EFP synthesis in OpMNPV-infected L. dispar cells were examined at various times throughout the infection cycle. Using pulse labeling and immunoprecipitation, GP64 EFP synthesis was detected within 2 hr p.i., and the maximal rate of synthesis was observed in the period of 24-26 hr p.i., a time coincident with the onset of high level production of budded virus in OpMNPV-infected L. dispar cells. To determine the oligomeric structure of GP64 EFP, a soluble form of OpMNPV GP64 EFP was produced and examined by a combination of gel filtration chromatography, nonreducing SDS-PAGE, and mass spectrometry. Oligomeric GP64 EFP was identified as a trimeric molecule, that migrates as two discrete bands on nonreducing SDS-PAGE. Pulse-chase studies, performed at both early (12 hr p.i.) and late (36 hr p.i.) stages of the infection cycle, showed that GP64 EFP oligomerization is complete within 15 min after synthesis. Efficiency of oligomerization however was relatively low, with less than 33% of the synthesized GP64 EFP converted to trimers. The majority of monomeric GP64 EFP remaining in the cell appeared to be degraded within 30 to 45 min after synthesis. Analysis of the kinetics of carbohydrate processing at early (12 hr p.i.) and late (36 hr p.i.) times postinfection showed that for both early and late phases of infection, carbohydrate was rapidly added, and processing began between 10 and 20 min after GP64 EFP synthesis. Although carbohydrate processing was completed within approximately 90 min after synthesis during the early phase, the same process required approximately 150 min during the late phase. Thus, carbohydrate processing appeared to become less efficient as infection progressed. These studies thus show that GP64 EFP undergoes a rapid but inefficient oligomerization step that results in a homotrimeric structure for GP64 EFP. While carbohydrate addition is rapid, carbohydrate processing requires prolonged periods of time (with half-times of 45 to 75 min) and appears to become less efficient during the late phase of the infection.