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

项目总结 正痘病毒家族的几个成员，包括天花(天花的起因)和猴痘， 对人类健康的严重威胁。其他成员对他们的自然宿主也同样严厉，包括皮肤萎缩症 (ECTV)，鼠痘的原因--一种与天花有许多相似之处的疾病。相当大的致命性 这些大型DNA病毒在很大程度上可归因于它们的许多蛋白质，这些蛋白质既能阻碍天生的DNA病毒，也能阻止 适应性主机防御。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，但对它们的研究仍然很少。高度同源的B22 除了减毒的牛痘病毒外，整个正痘病毒都有家族成员存在。 几个世纪以来，它一直被用作天花疫苗。我们在这份关于C15的探索性R21提案中重点介绍了 ECTV的B22家庭成员。C15的缺失在体内将病毒从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种新的特性： 1)C15有效地和选择性地抑制CD4+T细胞的激活，抑制免疫细胞的组装 Synapse。2)除了靶向获得性免疫外，C15还促进了病毒早在3天内的复制 感染后，反映了对先天免疫的抑制。值得注意的是，C15对RAG依赖和 -先天免疫的独立组成部分。根据我们的初步数据和既定的文献，我们 假设C15靶向的RAG非依赖性成分是NK细胞介导的细胞溶解，而RAG- 依赖成分是记忆性CD8+T细胞(Trm)的旁观者激活。我们进一步假设 连接这两种细胞类型的分子靶点是NKG2D，它是一种由NK细胞和 TRM和以前被证明在防御ECTV方面发挥了重要作用。取材于多年的 痘病毒经验和广泛的成熟和尖端技术，我们将测试这三个 三个独立但互补的目标中的假设。该项目的成果可以大大提高 对正痘病毒发病机制的了解，以及更广泛地说，有助于 病毒：主机相互作用。此外，我们预计成果将启动几个后续项目，包括：a) 将CD4+T细胞抑制纳入未来的机制研究，b)其他B22家族成员的检查 以及，c)用于潜在的宿主反应治疗调节的C15衍生物的开发。

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.