Structural basis for ligand promiscuity and high signaling activity of Kaposi’s Sarcoma-associated Herpesvirus-encoded GPCR

Kaposi's Sarcoma-associated Herpesvirus encodes ORF74, a viral G protein-coupled receptor homologous to CXCR2, which plays a crucial role in Kaposi's Sarcoma development through its high basal signaling activity. Our cryoEM analysis of ORF74 in ligand-free, BRIL-fused ligand-free, and CXCL1-Gitrimer-bound forms elucidated its ligand-independent signaling activity. The widely open, static extracellular cavity contributes to its ligand promiscuity by facilitating dynamic access and various binding modes. Impairment of CWxP, E/DRY, and NPxxY micro-switches destabilizes the inactive structure of ORF74 to allow ligand-independent activation. Metadynamics simulations demonstrate a highly dynamic equilibrium between inactive and active conformations in ORF74, enabling spontaneous inactive-active transitions. Finally, CXCR2-ORF74 chimeras underscore the functional importance of intracellular loops 2 and 3 in modulating basal and ligand-induced receptor activity. This study reveals the multifactorial structural elements of KSHV ORF74 that are responsible for its promiscuous ligand binding, spontaneous inactive-active conformational transitions, and ligand-independent high basal signaling, explaining the structural basis of ORF74-mediated viral oncogenesis.

卡波西肉瘤相关疱疹病毒（Kaposi's Sarcoma-associated Herpesvirus, KSHV）编码开放阅读框74（ORF74），这是一种与趋化因子受体2（CXCR2）同源的病毒G蛋白偶联受体（G protein-coupled receptor, GPCR），其高水平基础信号活性在卡波西肉瘤的发生发展中发挥关键作用。本研究针对配体游离态、融合布里奇曼溶菌酶（BRIL）的配体游离态，以及结合趋化因子配体1（CXCL1）与G蛋白三聚体（Gitrimer）的ORF74开展冷冻电镜（cryo-electron microscopy, cryoEM）分析，阐明了其配体非依赖型信号活性。该受体宽阔且静态的细胞外腔室可通过促进配体的动态接入与多样结合模式，加剧其配体混杂性。CWxP、E/DRY及NPxxY微开关的功能受损会破坏ORF74的非活性构象，使其能够发生配体非依赖型激活。元动力学模拟（Metadynamics simulations）结果显示，ORF74在非活性与活性构象间处于高度动态的平衡状态，可自发完成非活性-活性构象的转换。最后，通过CXCR2-ORF74嵌合受体实验，本研究证实了细胞内环2与3在调控受体基础活性及配体诱导活性中的功能性关键作用。本研究揭示了KSHV ORF74介导配体混杂结合、自发非活性-活性构象转换及配体非依赖型高基础信号转导的多维度结构基础，阐明了ORF74介导病毒致癌作用的结构机制。