Adaptive Mutations in the Nuclear Export Protein of Human-Derived H5N1 Strains Facilitate a Polymerase Activity-Enhancing Conformation
Adaptive Mutations in the Nuclear Export Protein of Human-Derived H5N1 Strains Facilitate a Polymerase Activity-Enhancing Conformation
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DOI：
10.1128/jvi.01495-13
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发表时间：
2013-10
影响因子：
5.4
通讯作者：
Peter Reuther;S. Giese;Veronika Götz;Normann Kilb;B. Mänz;Linda Brunotte;M. Schwemmle
中科院分区：
文献类型：
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作者：
Peter Reuther;S. Giese;Veronika Götz;Normann Kilb;B. Mänz;Linda Brunotte;M. Schwemmle
具有适应性突变M16 I的高致病性人源H5 N1毒株A/Thailand/1（KAN-1）/2004的核输出蛋白（NEP）（NS 2）以浓度依赖性方式极大地增强人细胞中的聚合酶活性。虽然低NEP水平增强聚合酶活性，但高水平是抑制性的。为了深入了解潜在的机制，我们分析了NEP缺失突变体在人类细胞中重建后对聚合酶活性的影响。这表明NEP的聚合酶增强功能存在于C-末端部分，并且去除最后三个氨基酸完全消除了该活性。此外，与全长NEP相比，单独的C-末端部分表现出显著更高的活性，并且似乎是去调节的，因为即使是最高浓度也不会导致聚合酶活性的抑制。为了确定顺式的N-和C-末端结构域之间的瞬时相互作用，我们将NEP的两端融合到分裂点击甲虫荧光素酶并进行片段互补测定。随着温度的降低，观察到荧光素酶活性增加，表明C-和N-末端结构域之间的分子内结合优先在低温下稳定。适应性突变M16 I或适应性突变的组合（M16 I、Y 41 C和E75 G）显著降低了这种稳定作用，这也在34°C下进一步增加了聚合酶活性。因此，我们提出了一个模型，其中NEP的N-末端部分发挥抑制功能的C-末端结构域的回折叠。在这个模型中，NEP中的适应性突变减少了C-和N-末端结构域之间的结合，从而允许蛋白质“打开”并在低温下变得有活性。
ABSTRACT The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase activity in human cells in a concentration-dependent manner. While low NEP levels enhance the polymerase activity, high levels are inhibitory. To gain insights into the underlying mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP resides in the C-terminal moiety and that removal of the last three amino acids completely abrogates this activity. Moreover, compared to full-length NEP, the C-terminal moiety alone exhibited significantly higher activity and seemed to be deregulated, since even the highest concentration did not result in an inhibition of polymerase activity. To determine transient interactions between the N- and C-terminal domains in cis, we fused both ends of NEP to a split click beetle luciferase and performed fragment complementation assays. With decreasing temperature, increased luciferase activity was observed, suggesting that intramolecular binding between the C- and N-terminal domains is preferentially stabilized at low temperatures. This stabilizing effect was significantly reduced with the adaptive mutation M16I or a combination of adaptive mutations (M16I, Y41C, and E75G), which further increased polymerase activity also at 34°C. We therefore propose a model in which the N-terminal moiety of NEP exerts an inhibitory function by back-folding to the C-terminal domain. In this model, adaptive mutations in NEP decrease binding between the C- and N-terminal domains, thereby allowing the protein to “open up” and become active already at a low temperature.