Kingsley D H, Behbahani A, Rashtian A, Blissard G W, Zimmerberg J
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-1855, USA.
Mol Biol Cell. 1999 Dec;10(12):4191-200. doi: 10.1091/mbc.10.12.4191.
Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH-induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.
病毒融合蛋白三聚体在限制膜融合中的脂质方面可能发挥关键作用。由于许多病毒融合蛋白的三聚体寡聚体通常由疏水的4-3七肽重复序列稳定，更高阶的寡聚体可能由类似序列稳定。苜蓿银纹夜蛾多粒包埋核型多角体病毒包膜糖蛋白GP64的假定寡聚化结构域附近存在一个疏水的4-3七肽重复序列。我们进行了诱变和肽抑制研究，以确定该序列是否可能在膜融合催化中发挥作用。首先，鉴定了疏水4-3七肽重复基序氨基末端及其侧翼内的亮氨酸到丙氨酸突变体，这些突变体寡聚形成三聚体并转运到昆虫Sf9细胞表面。根据构象敏感单克隆抗体的反应性判断，这些突变体在中性pH下保持其野生型构象，并在酸性条件下改变构象。然而，这些突变体在膜融合方面存在缺陷。其次，合成了编码GP64疏水4-3七肽重复序列侧翼部分的肽。添加该肽导致膜融合受到抑制，这种抑制仅在低pH作用期间存在该肽时发生。如通过构象敏感表位的丧失所确定的，在低pH作用期间存在该肽并不阻止低pH诱导的构象变化。在对照实验中，组成相同但序列不同的肽或编码埃博拉病毒GP七肽基序一部分的肽对GP64介导的融合没有影响。此外，当流感病毒的血凝素（X31株）融合蛋白在Sf9细胞中功能性表达时，未观察到对血凝素介导的融合有影响，这表明该肽对其他融合蛋白或细胞膜没有非特异性作用。总体而言，这些研究表明，与疏水4-3七肽重复序列相邻并包括其部分的GP64特定肽序列在膜融合中发挥动态作用，该阶段在蛋白质构象变化起始的下游但在脂质混合的上游。
