Molecular mechanism by which gamma-herpesviruses evade autophagy

描述（由申请人提供）：β-疱疹病毒科包括广泛分布的重要人类病原体，如EB病毒，其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，以及卡波西肉瘤病毒，其引起卡波西肉瘤肿瘤，特别是在免疫功能低下的个体如AIDS患者和移植受体中。3-疱疹病毒编码抗凋亡细胞Bcl-2蛋白（c-Bcl-2）的同源物，推测通过阻断细胞死亡促进病毒繁殖和致癌性。抗凋亡Bcl-2同系物防止凋亡的关键机制是通过隔离促凋亡Bcl-2同系物。虽然病毒Bcl-2（v-Bcl-2）和c-Bcl-2同源物具有非常低的序列同一性，但它们具有非常相似的三维结构。以前的结构，生物化学和诱变分析表明，抗凋亡Bcl-2同系物，包括大多数v-Bcl-2同系物的表面上的疏水沟，结合亲凋亡Bcl-2同系物的两亲性BH 3螺旋结构域。尽管有这种共同的结合BH 3结构域的一般机制，但不同的Bcl-2同源物对各种BH 3结构域显示出独特的特异性，这是由于它们的氨基酸序列不同。3-疱疹病毒还显示出抑制自噬，自噬是一种重要的细胞过程，通过该过程，细胞器、大蛋白质聚集体、稳定蛋白质以及细胞内病原体被封闭在双膜囊泡中并被靶向用于溶酶体降解。自噬通常由c-Bcl-2同源物与关键的自噬效应蛋白Beclin 1的相互作用调节，并且β-疱疹病毒对自噬的下调由v-Bcl-2同源物与Beclin 1的相互作用介导。最近，Beclin 1也被证明含有BH 3结构域，其构成与Bcl-XL和其他c-Bcl-2同源物结合的主要决定因素。我们的初步数据表明，Beclin 1与BH 3结构域相邻的其他区域也参与与Bcl-2同源物的相互作用。此外，各种c-和v-Bcl-2同源物结合到Beclin 1上的重叠但不相同的位点。这项资助的目的是描述Beclin 1与v-Bcl-2和c-Bcl-2同源物相互作用的差异，并验证相似性。相互作用将首先通过结合亲和力的生物化学测量来定量。结构测定和分析将提供每种相互作用的详细信息，确定关键特异性决定簇，这将通过定量所选突变体的结合亲和力来确认。最后，将测试ABT 737（一种抑制某些c-Bcl-2同系物和BH 3结构域结合的肽模拟物）破坏v-Bcl-2同系物和Beclin 1相互作用的能力。因此，这项研究将提供一个更好的理解疱疹病毒致病性的分子机制，自噬的细胞过程，疱疹病毒和宿主蛋白之间的相互作用，以及如何选择性地修改这些相互作用的诱变或小分子抑制剂，无论是为了进一步研究的目的，以及为设计潜在的治疗方法来治疗这一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，其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，以及KSHV，其引起卡波西肉瘤肿瘤，特别是在免疫受损个体如AIDS患者和移植受体中。这些病毒已经进化到下调自噬，这是一种已知可以降解许多病原体的宿主防御机制。本申请旨在研究3-疱疹病毒抑制自噬机制的原子细节，从而更好地理解3-疱疹病毒的致病性。

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.