The B22 family of orthopoxvirus virulence factors: Investigating structure/function of these potent, multifaceted immunoevasins

PROJECT SUMMARY Several members of the orthopoxvirus (OPXV) genus, including variola (the cause of smallpox), monkeypox virus (MPXV) and camelpox virus (CMPV) pose serious threats to human health, while the ultimate impact of emerging OPXVs of concern such as Alaskapox virus (AKPV) is unknown. Other OPXVs are highly pathogenic in their natural hosts, including ectromelia (ECTV), the cause of mousepox - a disease with many similarities to smallpox. It is concerning, then, that the extreme virulence typical of the OPXVs remains poorly understood. Central to OPXV virulence are the virus-encoded proteins that impede host defenses. This proposal focuses on members of the B22 family of OPXV immunoevasins due to their potency, complexity and limited characterization. With respect to potency, MPXV197 significantly enhances mortality in a nonhuman primate model, and ECTV C15 alone determines whether ECTV is 100% lethal or 100% survivable in a BALB/c mouse infection model. With respect to complexity, B22 family members are exceedingly large (~1900 aa), highly homologous multimembrane-spanning glycoproteins. We have observed that C15 is novel in targeting both adaptive and innate host responses, preventing activation of CD4+ and CD8+ T cells (TCD4 and TCD8), and interfering with the ability of natural killer (NK) cells to engage infected cells. Other B22 members have been implicated in hindering only TCD4 and/or TCD8 function; however, given the high degrees of homology, we suspect common functionalities, including targeting of NK cells. With respect to limited characterization, essentially no structure/function studies have been done with any B22 member. Through application of such studies to B22 members, key insights into OPXV virulence will be gained, potentially leading to novel therapeutic strategies and enhanced general principles of viral virulence. Guided by published and preliminary data, we propose a model in which topologically complex B22 members contain two functional subunits created by posttranslational cleavage: 1) an upstream (N-terminal) subunit that interferes with NK cell recognition via an MHC class I-like structure. 2) a C-terminal subunit that inhibits conjugation of TCD4 and TCD8 with their cognate targets. This model will be tested by pursuing three complementary aims that: 1) reveal the structural properties of B22 members, 2) identify and map the functional activities of B22 members, and 3) elucidate the viral and cellular binding partners of B22 proteins. The value of these studies derives from: a) the significance of many OPXV members as human pathogens, b) the potency of B22 members as virulence factors, c) the enigmatic immunoevasion activities of B22 members, and d) the potential for the proposed work to lead to new insights into virus:host interplay, the development of novel antiviral strategies and many future lines of investigation, including elucidation of the advantage to aggregating innate and adaptive immunoevasion activities into a single protein.