Institute of Molecular Genetics, National Research Council, Pavia, Italy.
PLoS One. 2011 May 12;6(5):e19810. doi: 10.1371/journal.pone.0019810.
DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.
DEAD -box 蛋白是一类具有核酸依赖性 ATP 酶、RNA 转位酶和解旋酶活性的酶。人类 DEAD-box 蛋白 DDX3 已被证明在肿瘤增殖和病毒感染中发挥重要作用。特别是，DDX3 已被确定为 HIV-1 复制的必需辅助因子。在这里，我们从核酸结合、ATP 酶和解旋酶活性等方面对一组 DDX3 突变体进行了生化特性分析。特别是，我们解决了 DDX3 基序 I 和 Ia 之间独特插入序列的功能作用，并提供了其与核酸结合和 HIV-1 复制相关的证据。我们表明，缺乏该结构域的人 DDX3 与 HIV-1 RNA 的结合亲和力较低。此外，通过噬菌体展示筛选出的针对该插入序列的特异性肽配体在转导到 HelaP4 细胞后会干扰 HIV-1 的复制。除了拓宽我们对这种重要蛋白质结构-功能关系的理解之外，我们的结果还确定了 DDX3 的一个特定结构域，该结构域可能适合作为设计用于抑制 HIV-1 复制的细胞辅助因子的抗病毒药物的靶标。
