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

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.