Nakashima Masaaki, Ode Hirotaka, Kawamura Takashi, Kitamura Shingo, Naganawa Yuriko, Awazu Hiroaki, Tsuzuki Shinya, Matsuoka Kazuhiro, Nemoto Michiko, Hachiya Atsuko, Sugiura Wataru, Yokomaku Yoshiyuki, Watanabe Nobuhisa, Iwatani Yasumasa
Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Aichi, Japan Department of Biotechnology, Nagoya University Graduate School of Engineering, Nagoya, Aichi, Japan.
Clinical Research Center, National Hospital Organization Nagoya Medical Center, Nagoya, Aichi, Japan.
J Virol. 2015 Nov 4;90(2):1034-47. doi: 10.1128/JVI.02369-15. Print 2016 Jan 15.
The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases.
HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.
HIV-1病毒感染因子（Vif）蛋白通过将细胞抗病毒胞苷脱氨酶载脂蛋白B mRNA编辑酶催化多肽样3F（APOBEC3F，A3F）特异性靶向蛋白酶体降解，从而使其在病毒感染细胞中失活。多项研究确定了参与A3F相互作用的Vif序列基序，而基于我们对高度相似的载脂蛋白B mRNA编辑酶催化多肽样3C（APOBEC3C，A3C）的分析，提出了一个Vif结合A3F界面。然而，Vif-A3F特异性识别的结构机制仍知之甚少。在此，我们报告了HIV-1 Vif和A3F分子相互作用界面的结构特征。对Vif进行丙氨酸扫描分析发现，位于保守的Vif F1-、F2-和F3-盒基序内的六个残基对于A3C和A3F的降解均至关重要，另外四个残基是A3F降解所特有的。基于HIV-1 Vif晶体结构对Vif结构进行建模显示，Vif F1-、F2-和F3-盒基序的三个不连续柔性环在空间上聚集形成一个柔性A3F相互作用界面，该界面呈现出疏水和亲水表面。我们发现，参与与A3C和A3F相互作用的碱性Vif界面区域（R17、E171和R173）有所不同。此外，我们对A3F C末端结构域的晶体结构测定和广泛的突变分析表明，A3F界面包含一个独特的酸性延伸区域（L291、A292、R293和E324），这对于Vif相互作用至关重要，表明与A3C界面相比，与Vif界面存在额外的静电互补。综上所述，这些发现为A3F-Vif相互作用机制提供了结构上的见解，这将为利用细胞胞苷脱氨酶开发新型抗HIV-1药物提供重要依据。
HIV-1 Vif靶向细胞抗病毒A3F酶进行降解。然而，特定A3F识别的结构机制细节仍不清楚。本研究报告了HIV-1 Vif和A3F分子相互作用界面的结构特征。Vif的三个不连续序列基序，即F1、F2和F3盒，组装形成一个A3F相互作用界面。此外，我们确定了负责Vif相互作用的野生型A3F C末端结构域的晶体结构。这些结果表明，静电和疏水相互作用都是驱动Vif-A3F结合的关键力量，并且Vif-A3F界面大于Vif-A3C界面。这些发现将使我们能够确定Vif-A3F复合物的构型并构建该复合物的结构模型，这将为抑制剂开发提供重要依据。
