Targeting of RAG-dependent and -independent innate immune responses by the Ectromelia C15 protein

项目摘要 正痘病毒家族的几个成员，包括天花（天花的原因）和猴痘， 严重威胁人类健康。其他成员在其自然宿主中也同样严重， （ECTV），鼠痘的原因-一种与天花有许多相似之处的疾病。相当大的毒力 这些大的DNA病毒在很大程度上归因于它们的许多蛋白质， 适应性宿主防御这些免疫调节蛋白中最大的是B22家族成员， 尽管它们的大小和对毒力的贡献，但仍然受到广泛的研究。高度同源B22 家族成员存在于除牛痘病毒（减毒的正痘病毒）之外的所有正痘病毒中 几个世纪以来一直被用作天花疫苗。我们在这个探索性的R21提案中关注C15， ECTV的B22家族成员C15的缺失使病毒在体内从100%致死转变为100%非致死 尽管对体外复制没有影响。我们对C15的初步研究揭示了两个新特性： 1)C15以抑制免疫球蛋白的组装的方式有效地和选择性地抑制CD 4 + T细胞活化。 突触2)除了靶向适应性免疫，C15还促进病毒复制早在3天 感染后，反映了先天免疫的抑制。值得注意的是，C15干扰RAG依赖性和 - 先天免疫的独立组成部分。根据我们的初步数据和现有文献，我们 假设C15靶向的RAG非依赖性组分是NK细胞介导的细胞溶解，RAG- 依赖性组分是记忆性CD 8 + T细胞的旁观者激活（Trm）。我们进一步假设， 连接这两种细胞类型的分子靶点是NKG 2D，一种由NK细胞表达的活化受体， Trm和以前显示在防御ECTV中发挥重要作用。从多年的 痘病毒的经验和广泛的既定和尖端技术，我们将测试这三个 三个独立但互补的目标。该项目的成果可以大大提高 了解正痘病毒的发病机制，更广泛地说，有助于 病毒：宿主相互作用。此外，我们预计成果将启动几个后续项目，包括： 在未来的机制研究中加入CD 4 + T细胞抑制，B）检查其他B22家族成员 和c）开发C15衍生物用于宿主反应的潜在治疗调节。

PROJECT SUMMARY Several members of the orthopoxvirus family, including variola (the cause of smallpox) and monkeypox, pose serious threats to human health. Other members are equally severe in their natural hosts, including ectromelia (ECTV), the cause of mousepox - a disease with many similarities to smallpox. The considerable virulence of these large DNA viruses is attributable in great measure to their many proteins that impede both innate and adaptive host defenses. The largest among these immunomodulatory proteins are the B22 family members, which, despite their size and contributions to virulence, remain vastly understudied. Highly homologous B22 family members are present throughout the orthopoxviruses except for vaccinia, the attenuated orthopoxvirus that has served as the smallpox vaccine for centuries. We focus in this exploratory R21 proposal on C15, the B22 family member of ECTV. Deletion of C15 converts the virus from 100% lethal to 100% nonlethal in vivo despite having no impact on replication in vitro. Our preliminary work with C15 has revealed two novel properties: 1) C15 potently and selectively inhibits CD4+ T cell activation in a way that inhibits assembly of the immunological synapse. 2) In addition to targeting adaptive immunity, C15 also facilitates viral replication as early as 3 days post infection, reflecting inhibition of innate immunity. Remarkably, C15 interferes with both RAG-dependent and -independent components of innate immunity. Based on our preliminary data and the established literature, we hypothesize that the RAG-independent component targeted by C15 is NK cell-mediated cytolysis and the RAG- dependent component is bystander activation of memory CD8+ T cells (Trm). We further hypothesize that the molecular target linking these two cell types is NKG2D, an activating receptor expressed by both NK cells and Trm and shown previously to play an important role in defense against ECTV. Drawing from many years of poxvirus experience and a wide range of established and cutting-edge techniques, we will test these three hypotheses in three independent but complementary aims. Outcomes of this project could considerably enhance understanding of orthopoxvirus pathogenesis and, more broadly, contribute to fundamental principles of virus:host interplay. In addition, we anticipate that results will launch several subsequent projects including: a) incorporation of CD4+ T cell inhibition in future mechanistic studies, b) examination of other B22 family members and, c) the development of C15 derivatives for potential therapeutic modulation of host responses.