Molecular mechanism by which gamma-herpesviruses evade autophagy

描述(申请人提供)：3-疱疹病毒科包括广泛的、重要的人类病原体，如导致传染性单核细胞增多症和淋巴和上皮组织恶性肿瘤的爱泼斯坦-巴尔病毒，以及导致卡波西肉瘤的卡波西肉瘤病毒，特别是在免疫功能受损的人中，如艾滋病患者和移植受者。3-疱疹病毒编码抗细胞凋亡的Bcl2蛋白(c-Bcl2)的同源物，推测通过阻止细胞死亡来促进病毒的繁殖和致癌作用。抗凋亡的Bcl2同源物阻止细胞凋亡的一个关键机制是隔离促凋亡的Bcl2同源物。虽然病毒Bcl2(v-Bcl2)和c-Bcl2同源物的序列同源性很低，但它们具有非常相似的三维结构。以往的结构、生化和诱变分析表明，抗凋亡的Bcl2同源物，包括大多数v-Bcl2同源物，表面都有疏水凹槽与促凋亡的BH3同源物的两亲性螺旋结构域结合。尽管有这种共同的结合BH3结构域的一般机制，但不同的Bcl-2同源物对不同的BH3结构域表现出独特的特异性，这是由于它们的氨基酸序列不同。3-疱疹病毒也被证明抑制自噬，这是一个重要的细胞过程，细胞器、大的蛋白质聚集体、稳定的蛋白质以及细胞内的病原体被包裹在双层膜小泡中，并以溶酶体降解为目标。自噬通常通过c-Bcl2同源蛋白与关键的自噬效应蛋白Beclin 1的相互作用来调节，而3-疱疹病毒对自噬的下调是通过v-Bcl2同源蛋白与Beclin 1的相互作用来介导的。最近，Beclin 1还被发现含有一个BH3结构域，它构成了与Bclxl和其他c-Bcl2同源蛋白结合的主要决定因素。我们的初步数据表明，Beclin 1与BH3结构域相邻的其他区域也参与了与Bcl-2同源物的相互作用。此外，不同的c-和v-bcl2同源物与Beclin 1上的重叠但不完全相同的位点结合。本研究的目的是表征Beclin 1与v-bcl2和c-bcl2同源物相互作用中的差异，并验证其相似性。相互作用将首先通过结合亲和力的生化测量来量化。结构确定和分析将提供每个相互作用的细节，确定关键的特异性决定因素，这将通过量化选定突变体的结合亲和力来确认。最后，ABT737，一种抑制某些c-Bcl-2同源物和BH3结构域结合的多肽模拟物，将被测试其破坏v-Bcl-2同源物与Beclin 1相互作用的能力。因此，这项研究将提供更好地理解疱疹病毒致病的分子机制，自噬的细胞过程，疱疹病毒与宿主蛋白之间的相互作用，以及如何通过突变或小分子抑制剂选择性地改变这些相互作用的信息，以便进一步研究以及设计潜在的治疗方法来治疗这类医学上重要的病原体。公共卫生相关性：重要的3-疱疹病毒人类病原体包括EBV和KSHV，EBV可引起传染性单核细胞增多症和淋巴和上皮组织的恶性肿瘤，KSHV可引起卡波西肉瘤，尤其是在免疫功能低下的人中，如艾滋病患者和移植受者。这些病毒已经进化到下调自噬，自噬是一种已知能降解大量病原体的宿主防御机制。这项应用旨在研究3-疱疹病毒抑制自噬机制的原子细节，为更好地了解3-疱疹病毒的致病性提供依据。

DESCRIPTION (provided by applicant): The 3-herpesviridae includes widespread, important human pathogens like Epstein-Barr virus which causes infectious mononucleosis and malignant tumors of lymphoid and epithelial tissues and Kaposi Sarcoma virus, which causes Kaposi sarcoma tumors, especially among immunocompromised individuals such as AIDS patients and transplant recipients. 3-Herpesviruses encode homologs of the anti-apoptotic, cellular Bcl-2 protein (c-Bcl-2), presumably to facilitate viral propagation and oncogenicity by blocking cell death. A key mechanism by which anti-apoptotic Bcl-2 homologs prevent apoptosis is by sequestering pro-apoptotic Bcl-2 homologs. Although viral Bcl-2 (v-Bcl-2) and c-Bcl-2 homologs share very low sequence identity, they have very similar three-dimensional structures. Previous structural, biochemical and mutagenic analyses showed that a hydrophobic groove on the surface of anti-apoptotic Bcl-2 homologs, including most v-Bcl-2 homologs, binds the amphipathic BH3 helical domains of pro-apoptotic Bcl-2 homologs. Despite this shared general mechanism for binding BH3 domains, the different Bcl-2 homologs show unique specificity toward various BH3 domains, arising from differences in their amino acid sequences. 3-herpesviruses have been shown to also inhibit autophagy, a vital cellular process by which organelles, large protein aggregates, stable proteins, as well as intracellular pathogens are enclosed in double-membrane vesicles and targeted for lysosomal degradation. Autophagy is normally modulated by the interaction of c-Bcl-2 homologs with a key autophagy effector protein, Beclin 1, and the down-regulation of autophagy by 3-herpesviruses is mediated by the interaction of v-Bcl-2 homologs with Beclin 1. Recently, Beclin 1 was also shown to contain a BH3 domain that constitutes the primary determinant of binding to Bcl-XL and other c-Bcl-2 homologs. Our preliminary data indicates that additional regions of Beclin 1 contiguous with the BH3 domain are also involved in interaction with Bcl-2 homologs. Further, the various c- and v-Bcl-2 homologs bind to overlapping, but not identical, sites on Beclin 1. The goal of this grant is to characterize the differences and also to verify the similarities, in the interaction of Beclin 1 with v-Bcl-2 and c-Bcl-2 homologs. Interactions will first be quantified by biochemical measurements of binding affinity. Structure determination and analyses will provide details of each interaction, identifying key specificity determinants, which will be confirmed by quantifying binding affinity of selected mutants. Finally, ABT737, a peptido-mimetic that inhibits binding of certain c-Bcl-2 homologs and BH3 domains, will be tested for its ability to disrupt the interaction of v-Bcl-2 homologs and Beclin 1. Thus, this research will provide a better understanding of the molecular mechanisms of herpesvirus pathogenicity, the cellular process of autophagy, the interaction between herpesviral and host proteins, and information on how to selectively modify these interactions either by mutagenesis or by small molecule inhibitors, both for the purposes of further research as well as for designing potential therapeutics to treat this medically important group of pathogens. PUBLIC HEALTH RELEVANCE: Important 3-Herpesviruses human pathogens include EBV, which causes infectious mononucleosis and malignant tumors of lymphoid and epithelial tissues, and KSHV, which causes Kaposi sarcoma tumors, especially among immuno-compromised individuals such as AIDS patients and transplant recipients. These viruses have evolved to down-regulate autophagy, a host defense mechanism known to degrade numerous pathogens. This application aims to investigate atomic details of the mechanism by which 3-herpesviruses inhibit autophagy, providing a better understanding of 3-herpesviral pathogenicity.