Functional dissection of the major structural protein of bluetongue virus: Identification of key residues within VP7 essential for capsid assembly
Functional dissection of the major structural protein of bluetongue virus: Identification of key residues within VP7 essential for capsid assembly
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DOI：
10.1128/jvi.74.18.8658-8669.2000
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发表时间：
2000-09-01
影响因子：
5.4
通讯作者：
Roy, P
中科院分区：
文献类型：
--
作者：
Limn, CK;Staeuber, N;Roy, P
VP 7三聚体的晶格形成二十面体蓝舌病毒（BTV）核心的表面。为了研究VP 7寡聚化在核心组装中的作用，基于BTV 10型VP 7分子的晶体结构，预测了一系列用于替换的残基，这些残基针对三聚体内的单体-单体接触。已经使用cDNA克隆创建了VP 7的7个位点特异性替换突变，并用于产生7种重组杆状病毒。研究了这些突变对VP 7溶解性、三聚化能力和在支架VP 3蛋白存在下形成核心样颗粒（CLP）的影响。在检测的7个VP 7突变体中，3个严重影响CLP的稳定性，而另外2个突变体对CLP稳定性的影响较小。只有一个突变体对稳定衣壳的形成没有明显的影响，其中271位残基（下结构域螺旋6）的保守酪氨酸被精氨酸取代的一个突变体形成不溶性聚集体，这意味着分子折叠的影响，尽管预测这种变化将被容纳。纯化所有六种可溶性VP 7突变体，并检查它们的三聚化能力。所有的突变体，包括那些没有形成稳定的CLP，组装成稳定的三聚体，这意味着单一的取代可能不足以扰乱复杂的单体-单体接触，虽然VP 7三聚体内的细微变化可能会破坏核心。这项研究强调了一些对BTV核心组装至关重要的关键残基，并说明了VP 7的结构如何在生物水平上低估了组装过程的动态性质。
A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer, Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid, One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.