Oomens A G, Blissard G W
Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, New York, 14853, USA.
Virology. 1999 Feb 15;254(2):297-314. doi: 10.1006/viro.1998.9523.
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 virus 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 vAc64- were pulse labeled, and progeny BV were isolated on equilibrium sucrose gradients and quantified. BV production from vAc64- was reduced to approximately 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 ( approximately 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 approximately 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.
苜蓿银纹夜蛾多粒包埋核型多角体病毒（AcMNPV）的出芽病毒粒子（BV）含有一种主要的包膜糖蛋白（GP64），该蛋白存在于受感染细胞的质膜上。GP64在病毒粒子通过质膜出芽的过程中被获取，这是病毒粒子在细胞表面组装的最后一步。先前的研究（S. A. Monsma、A. G. P. Oomens和G. W. Blissard（1996年）。《病毒学杂志》70卷，4607 - 4616页）表明，AcMNPV gp64基因的插入失活导致一种病毒无法在细胞间传播，且对经口感染的粉纹夜蛾幼虫无致死性。为了确定GP64是否参与病毒粒子出芽，我们测量了感染gp64缺失病毒的Sf9细胞产生的BV。用gp64缺失病毒vAc64 - 感染Sf9细胞，进行脉冲标记，然后在平衡蔗糖梯度上分离子代BV并进行定量。vAc64 - 产生的BV减少到野生型AcMNPV产生量的约2%。因此，GP64蛋白对高效的病毒粒子出芽很重要。为了确定GP64高度带电荷的7个氨基酸的胞质尾域（CTD）是否是病毒粒子产生所必需的，我们构建了一系列含有C末端截短或替换的GP64构建体。在转染细胞和野生型gp64基因被修饰的gp64取代的重组病毒中分析修饰后的GP64形式。从CTD删除1 - 7个氨基酸不影响GP64三聚体化、蛋白质转运到细胞表面或膜融合活性。然而，删除11或14个氨基酸，这去除了CTD和部分预测的跨膜（TM）域，虽能三聚体化，但由于这些截短蛋白的脱落，在细胞表面的水平较低。比较表达野生型或突变型GP64蛋白的重组病毒的生长曲线和标记子代BV产生的定量测量结果表明，删除7个残基的CTD仅适度降低感染性病毒粒子的产生（约50%）。然而，删除C末端11或14个氨基酸有更显著的影响。去除C末端11个氨基酸使感染性病毒滴度降低78 - 96%，标记子代病毒粒子减少91 - 92%。从C末端去除14个氨基酸导致子代BV减少约98%，并且产生一种显然无法在细胞培养中有效增殖的病毒。因此，GP64 CTD对于感染性BV的产生不是必需的，但去除CTD会导致出芽效率有可测量的降低。删除CTD加上跨膜域的小部分导致GP64脱落、表面水平降低以及BV产生显著减少。总之，这些数据表明GP64是BV产生中的一个重要且限制性的因素。
