APOBEC3 STRUCTURAL STUDIES

 描述(申请人提供)：抗病毒APOBEC3酶的发现被认为是艾滋病毒/艾滋病分子病毒学中最有希望的治疗突破之一。几种APOBEC3具有限制HIV复制的潜力，它们被整合到组装的病毒颗粒中，物理上干扰了逆转录的进程，并将病毒的cDNA胞嘧啶脱氨基为尿嘧啶。后者的抗病毒活性是APOBEC3介导的限制的明确标志，解释了在患者来源的病毒序列中经常观察到的基因组链G-to-A突变。然而，这些强大的抗病毒活性被HIV病毒粒子感染性因子(VIF)所抵消，VIF与CBF-β异源二聚体形成E3泛素连接酶复合体，降解APOBEC3酶。在这里，我们将解决这一领域的两个长期存在的问题。首先，我们将确定APOBEC3结合的Vif/CBF-β泛素连接酶复合体的X射线结构。这些结构将为APOBEC3-Vif结合机制、构象变化以及这些宿主-病原体复合体的整体组织提供见解。其次，我们将阐明APOBEC3/单链DNA复合体的X射线结构。这些结构将对酶催化、DNA底物识别以及DNA结合和催化活性释放之间的耦合机制提供深入的了解。我们预计，从长远来看，来自APOBEC3/Vif/CBF-beta和APOBEC3/单链DNA大分子复合体的结构信息将是重要的，因为该领域继续朝着抑制这些相互作用和通过天然免疫实现有效的HIV-1限制的方向发展。

 DESCRIPTION (provided by applicant): The discovery of the antiviral APOBEC3 enzymes is regarded as one of the most therapeutically promising breakthroughs in HIV/AIDS molecular virology. Several APOBEC3s have the potential to restrict HIV replication by incorporating into assembling viral particles, physically interfering with the progression of reverse transcription, and deaminating viral cDNA cytosines to uracils. The latter antiviral activity is the defining hallmark of APOBEC3-mediated restriction, explaining the genomic strand G-to-A mutations that are frequently observed in patient-derived viral sequences. However, these potent antiviral activities are counteracted by the HIV virion infectivity factor (Vif), which heterodimerizes with CBF-beta in order to form an E3 ubiquitin ligase complex that degrades APOBEC3 enzymes. Here, we will address two persisting problems in this field. First, we will determine x-ray structures of APOBEC3-bound Vif/CBF-beta ubiquitin ligase complexes. These structures will provide insights into the APOBEC3-Vif binding mechanism, conformational changes, and the overall organization of these host-pathogen complexes. Second, we will elucidate X-ray structures of APOBEC3/single-stranded DNA complexes. These structures will provide insights into the mechanisms of enzymatic catalysis, DNA substrate recognition, and coupling between DNA-binding and the release of catalytic activity. We anticipate that structural information from both APOBEC3/Vif/CBF-beta and APOBEC3/single-stranded DNA macromolecular complexes will be important in the longer-term as the field continues to move toward drugging these interactions and enabling potent HIV-1 restriction through natural innate immunity.