Molecular Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-1892, USA.
Biochemistry. 2010 Mar 23;49(11):2443-53. doi: 10.1021/bi902130j.
Cytomegaloviruses (CMVs) are ubiquitous species-specific viruses that establish acute, persistent, and latent infections. Both human and mouse CMVs encode proteins that inhibit the activation of natural killer (NK) cells by downregulating cellular ligands for the NK cell activating receptor, NKG2D. The MCMV glycoprotein m152/gp40 downregulates the surface expression of RAE-1 to prevent NK cell control in vivo. So far, it is unclear if there is a direct interaction between m152 and RAE-1 and, if so, if m152 interacts differentially with the five identified RAE-1 isoforms, which are expressed as two groups in MCMV-susceptible or -resistant mouse strains. To address these questions, we expressed and purified the extracellular domains of RAE-1 and m152 and performed size exclusion chromatography binding assays as well as analytical ultracentrifugation and isothermal titration calorimetry to characterize these interactions quantitatively. We further evaluated the role of full-length and naturally glycosylated m152 and RAE-1 in cotransfected HEK293T cells. Our results confirmed that m152 binds RAE-1 directly, relatively tightly (K(d) < 5 microM), and with 1:1 stoichiometry. The binding is quantitatively different depending on particular RAE-1 isoforms, corresponding to the susceptibility to downregulation by m152. A PLWY motif found in RAE-1beta, although contributing to its affinity for m152, does not influence the affinity of RAE-1gamma or RAE-1delta, suggesting that other differences contribute to the RAE-1-m152 interaction. Molecular modeling of the different RAE-1 isoforms suggests a potential site for the m152 interaction.
巨细胞病毒（CMV）是无处不在的种特异性病毒，可引起急性、持续性和潜伏性感染。人巨细胞病毒和鼠巨细胞病毒均编码可抑制自然杀伤（NK）细胞激活的蛋白，其通过下调 NK 细胞激活受体 NKG2D 的细胞配体来实现。MCMV 糖蛋白 m152/gp40 通过下调 RAE-1 的表面表达来防止体内 NK 细胞的控制。到目前为止，尚不清楚 m152 是否与 RAE-1 存在直接相互作用，如果存在，m152 是否与五个已鉴定的 RAE-1 同工型以不同的方式相互作用，这在 MCMV 易感或抗性小鼠品系中以两个组的形式表达。为了解决这些问题，我们表达并纯化了 RAE-1 和 m152 的细胞外结构域，并进行了排阻色谱结合测定以及分析超速离心和等温滴定量热法，以定量表征这些相互作用。我们进一步评估了全长和天然糖基化的 m152 和 RAE-1 在共转染的 HEK293T 细胞中的作用。我们的结果证实 m152 与 RAE-1 直接结合，亲和力较强（K(d)<5μM），且为 1:1 化学计量比。结合具有定量差异，具体取决于特定的 RAE-1 同工型，与 m152 的下调能力相对应。在 RAE-1beta 中发现的 PLWY 基序虽然有助于其与 m152 的亲和力，但不影响 RAE-1gamma 或 RAE-1delta 的亲和力，这表明其他差异有助于 RAE-1-m152 相互作用。不同 RAE-1 同工型的分子建模表明了 m152 相互作用的潜在位点。
