Requirement for GP64 to drive efficient budding of Autographa californica multicapsid nucleopolyhedrovirus
Requirement for GP64 to drive efficient budding of Autographa californica multicapsid nucleopolyhedrovirus
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DOI：
10.1006/viro.1998.9523
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发表时间：
1999-02-15
期刊：
影响因子：
3.7
通讯作者：
Blissard, GW
中科院分区：
文献类型：
--
作者：
Oomens, AGP;Blissard, GW
苜蓿银纹夜蛾核型多角体病毒（Autographa californica multicapsid nucleopolyhedrovirus，AcMNPV）的芽状病毒粒子（Budded virion，BV）含有一个主要的包膜糖蛋白（GP 64），它存在于被感染细胞的质膜上。GP 64在通过质膜的出芽过程中被病毒体获得，这是出芽病毒体在细胞表面组装的最后一步。以前的研究（S。A. Monsma，A. G. P. Oomens和G. W. Blissard（1996年）。J Virol。70，4607-4616）表明AcMNPV gp 64基因的插入失活导致罗斯不能从一个细胞移动到另一个细胞，并且对经口感染的粉纹夜蛾幼虫是非致死的。为了确定GP 64是否参与病毒体出芽，我们测量了用gp 64无效病毒感染的Sf 9细胞的BV产生。用gp 64空病毒vAC（64-）感染的Sf 9细胞进行脉冲标记，并在平衡蔗糖梯度上分离子代BV并定量。vAC（64-）的BV产量降低至野生型AcMNPV的2%。因此，GP 64蛋白对于有效的病毒体出芽是重要的。为了确定GP 64的高度带电的7-氨基酸胞质尾域（CTD）是否是病毒体产生所需的，我们产生了一系列含有C-末端截短或取代的GP 64构建体。在转染的细胞和重组病毒中分析GP 64的修饰形式，其中野生型gp 64基因被修饰的gp 64取代。从CTD中删除1-7个氨基酸不影响GP 64三聚体、蛋白质转运至细胞表面或膜融合活性。然而，11或14个氨基酸的缺失，去除CTD和预测的跨膜（TM）结构域的部分，是三聚化的，但由于这些截短的蛋白质的脱落，在细胞表面上以较低的水平存在。生长曲线的比较和标记的子代BV生产的重组病毒表达野生型或突变体GP 64蛋白的定量测量表明，删除7个残基的CTD仅适度减少感染性病毒体的生产（类似于50%）。然而，C末端11或14个氨基酸的缺失具有更实质性的影响。去除C末端11个氨基酸使感染性病毒的滴度降低78-96%，并且标记的子代病毒体降低91- 92%。从C末端去除14个氨基酸导致子代BV和明显不能在细胞培养物中有效繁殖的病毒减少类似于98%。因此，GP 64 CTD对于感染性BV的产生不是必需的，但是CTD的去除导致出芽效率的可测量的降低。CTD加上跨膜结构域的一小部分的缺失导致GP 64脱落，表面水平降低，BV产生急剧减少。总之，这些数据表明GP 64是BV产生的重要限制因素。(C)北京：科学出版社.
Budded virions (BV) of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein (GP64) that is present on the plasma membrane of infected cells. GP64 is acquired by virions during budding through the plasma membrane, the final step in assembly of the budded virion at the cell surface. Previous studies (S. A. Monsma, A. G. P. Oomens, and G. W. Blissard (1996). J Virol. 70, 4607-4616) showed that insertional inactivation of the AcMNPV gp64 gene resulted in a vi rus unable to move from cell to cell and nonlethal to orally infected Trichoplusia ni larvae. To determine whether GP64 is involved in virion budding, we measured BV production from Sf9 cells infected with a gp64null virus. Sf9 cells infected with gp64null virus vAC(64-) were pulse labeled, and progeny BV were isolated on equilibrium sucrose gradients and quantified. BV production from vAC(64-) was reduced to similar to 2% of that from wild-type AcMNPV. Thus the GP64 protein is important for efficient virion budding. To determine whether the highly charged 7-amino acid cytoplasmic tail domain (CTD) of GP64 was required for virion production, we generated a series of GP64 constructs containing C-terminal truncations or substitutions. Modified forms of GP64 were analyzed in transfected cells and in recombinant viruses in which the wild-type gp64 gene was replaced with a modified gp64. Deletion of 1-7 amino acids from the CTD did not affect GP64 trimerization, protein transport to the cell surface, or membrane fusion activity. However, deletions of 11 or 14 amino acids, which removed the CTD and portions of the predicted transmembrane (TM) domain, were trimerized but were present at lower levels on the cell surface due to shedding of these truncated proteins. Comparisons of growth curves and quantitative measurements of labeled progeny BV production from recombinant viruses expressing either wild-type or mutant GP64 proteins showed that deletion of the 7-residue CTD only moderately reduced the production of infectious virions (similar to 50%). However, deletions of the C terminal 11 or 14 amino acids had more substantial effects. Removal of the C terminal 11 amino acids reduced titers of infectious virus by 78-96% and labeled progeny virions were reduced by 91-92%. Removal of 14 amino acids from the C terminus resulted in an similar to 98% reduction in progeny BV and a virus that was apparently incapable of efficient propagation in cell culture. Thus the GP64 CTD is not essential for production of infectious BV, but removal of the CTD results in a measurable reduction in budding efficiency. Deletion of the CTD plus small portions of the transmembrane domain resulted in shedding of GP64, reduced surface levels, and a dramatic reduction in the production of BV. Together, these data indicate that GP64 is an important and limiting factor in BV production. (C) 1999 Academic Press.