Paterson R G, Russell C J, Lamb R A
Department of Biochemistry, Molecular Biology, and Cell Biology, Evanston, IL 60208-3500, USA.
Virology. 2000 Apr 25;270(1):17-30. doi: 10.1006/viro.2000.0267.
The fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37 degrees C, and does not cause fusion at 37 degrees C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309-319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state.
副粘病毒SV5株W3A的融合（F）蛋白在不共表达SV5血凝素神经氨酸酶（HN）糖蛋白的情况下即可导致合胞体形成，而SV5株WR的F蛋白则需要与HN共表达才能具有融合活性。SV5株W3A和WR在第22、443和516位氨基酸残基处存在三个氨基酸差异。将W3A F蛋白的P22、S443和V516残基分别替换为WR F蛋白中的氨基酸（分别为L22、P443和A516）。构建、表达了三个单突变体、三个双突变体和三突变体，并使用三种不同的检测方法对其融合活性进行了检测。突变体P22L在生理条件下不引起融合，但在升高温度时融合被激活。与W3A F蛋白相比，突变体S443P以更快的速率和更大的程度增强了融合动力学，且具有更低的激活温度。突变体V516A对F蛋白介导的融合影响很小。双突变体P22L,S443P能够引起融合，这表明这两个突变对融合激活具有相反的作用。WR F蛋白需要与HN共表达才能在37℃时引起融合，当与流感病毒血凝素（HA）共表达时在37℃时不引起融合；然而，在升高温度时，WR F蛋白与HA共表达会导致融合激活。在SV5 F蛋白核心三聚体的晶体结构中（Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309 - 319），S443是延伸链区域中的最后一个残基（具有可解释的电子密度），且S443的温度因子较高，表明该位点存在构象灵活性。因此，第22和443位残基处脯氨酸的存在可能会使F蛋白不稳定，从而降低触发假定的向融合活性状态构象变化所需的能量。
