Viral oncoproteins: Revealing novel structural motifs to target tumor suppressors

描述（由申请人提供）：该提案将揭示使微小病毒癌蛋白能够靶向并破坏多种关键肿瘤抑制途径的结构机制和基序。最终目标是利用这些知识开发针对难治性肿瘤靶点的新型病毒疗法。最小DNA肿瘤病毒基因组的进化选择了小的病毒癌蛋白，这些蛋白劫持了关键的细胞蛋白相互作用网络，这些网络也是癌症突变的目标。然而，小腺病毒癌蛋白的主要相互作用的结构基础仍然难以捉摸，因为它们的完整结构都没有得到解决。这代表了对腺病毒生物学和使小病毒蛋白质“获胜”的结构原理的理解的根本差距。为了解决这个问题，在2.1 °处解析了E4-ORF 3二聚体的结构。E4-ORF 3是一种13 kDa的蛋白质，其组装核聚合物网络，该网络结合并破坏PML、TRIM 24和MRE 11/RAD 50/NBS 1（MRN）肿瘤抑制复合物。此外，E4-ORF 3通过未知的机制诱导p53靶基因和抗病毒基因的异染色质沉默。与典型的腺病毒癌蛋白E1 A相反，E4-ORF 3具有离散的有序结构，并且不是任何已知的细胞聚合物或癌基因的结构同源物。E4-ORF 3形成具有中心β-核心的二聚体亚基，其通过其C-末端尾部的相互和非相互交换进一步共组装。E4-ORF 3的高阶组装是创建亲合力驱动的与PML的相互作用和紧急MRN结合界面所必需的。这项研究建立在这些研究的基础上。目标1将揭示驱动野生型E4-ORF 3组装的结构和更高级的寡聚相互作用，并且是其破坏多种肿瘤抑制因子以促进病毒复制的功能所必需的。目的2将利用E4-ORF 3的结构作为合理的基础来鉴定靶向PML、MRN和TRIM 24肿瘤抑制复合物的新结构基序，这些复合物是重要的治疗靶点。离散的E4-ORF 3突变将被工程化，选择性地解偶联其与不同肿瘤抑制复合物的相互作用，以揭示它们在病毒感染中的各自贡献。这将为开发新的选择性地在具有特定肿瘤抑制途径突变的肿瘤细胞中复制的恶性肿瘤疗法提供合理的基础。E4-ORF 3二聚化产生了一种新的结合裂缝，决定了它在细胞核中的组装位点，并且是沉默p53靶基因所必需的。目标3将使用 病毒工程和综合比较基因组学方法的组合，以确定裂缝内的残基是否将E4-ORF 3组装靶向特定的基因组位点，在那里它与H10中的基序结合，并诱导p53和抗病毒基因的抑制性异染色质沉默。这将揭示在感染中沉默p53的新靶点和机制，并且也可能被癌症突变破坏。

DESCRIPTION (provided by applicant): This proposal will reveal the structural mechanisms and motifs that enable a tiny viral oncoprotein to target and disrupt multiple critical tumor suppressor pathways. The ultimate goal is to exploit this knowledge to develop novel viral therapies against intractable tumor targets. The evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction network that are also targeted by mutations in cancer. However, the structural basis for the dominant interactions of small adenovirus oncoproteins has remained elusive, as none of their complete structures have been solved. This represents a fundamental gap in the understanding of Adenovirus biology and the structural principles that enable small viral proteins to 'win'. To address this the structure of an E4-ORF3 dimer at 2.1¿ was solved. E4-ORF3 is a 13kDa protein that assembles a nuclear polymer network that binds and disrupts the PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressor complexes. In addition, E4-ORF3 induces heterochromatin silencing at p53 target genes and anti-viral genes through an unknown mechanism. In contrast to the archetypal Adenovirus oncoprotein, E1A, E4-ORF3 has a discrete ordered structure and is not a structural homologue of any known cellular polymers or oncogenes. E4-ORF3 forms dimer subunits with a central beta-core that further co-assemble through reciprocal and non-reciprocal exchanges of their C-terminal tails. The higher order assembly of E4-ORF3 is required for creating avidity-driven interactions with PML and an emergent MRN binding interface. This proposed research builds on these studies. Aim 1 will reveal the structure and higher order oligomeric interactions that drive the assembly of wild type E4-ORF3 and are required for its functions in disrupting multiple tumor suppressors to facilitate viral replication. Aim 2 will use the structure of E4-ORF3 as a rational basis to identify new structural motifs that target the PML, MRN and TRIM24 tumor suppressor complexes, which are important therapeutic targets. Discrete E4-ORF3 mutations will be engineered that selectively uncouple its interactions with different tumor suppressor complexes to reveal their respective contributions in viral infection. This will provide a rational basis for the development of novel vral cancer therapies that selectively replicate in tumor cells with particular tumor suppressor pathway mutations. E4-ORF3 dimerization creates a novel binding-cleft that determines the sites of its assembly in the nucleus and is required for silencing p53 target genes. Aim 3 will use a combination of viral engineering and integrative comparative genomics approaches to determine if residues within the cleft target the E4-ORF3 assembly to specific genomic loci where it binds to a motif in H10 and to induce repressive heterochromatin silencing of p53 and anti-viral genes. This will reveal new targets and mechanisms that silence p53 in infection and that could also be disrupted by mutations in cancer.