Uncovering the molecular underpinnings of C15, a potent orthopoxvirus virulence factor

PROJECT SUMMARY The orthopoxvirus (OPXV) genus is home to many severe mammalian pathogens, including variola (VARV) and Mpox (MPXV). Though VARV was eradicated in 1980, OPXVs remain a significant public health threat; the recent worldwide outbreak of MPXV has highlighted the susceptibility of individuals to OPXV infection, the sub-optimal efficacy of current FDA-approved vaccines, and the limited options to treat active infections. These concerns are amplified by the fact that the pathogenesis of OPXVs remains poorly understood. In principle, OPXV virulence is attributed to the myriad of 'immunoevasins' they encode. However, many of these proteins do not have well-defined functions. Given the critical need for more robust strategies to combat OPXV infection, it is essential to define the major determinants of OPXV virulence. One promising avenue of study focuses on the B22 protein family, which comprises a group of glycoproteins that are conserved across all pathogenic OPXVs. In ectromelia (ECTV), the cause of mousepox, we found that its B22 family member, C15, is essential for mortality and pathogenesis in mice. Functionally, our lab has demonstrated that C15 potently inhibits both natural killer (NK) cell-mediated control and T cell activation during ECTV infection, highlighting the capacity for C15 to target both the innate and adaptive immune response. However, much of the underlying cell biology of C15 (and B22 proteins by extension) as well as the structural basis for its activity remain unknown, limiting potential as a therapeutic target. This proposal, composed of two aims, outlines an investigative approach to define the molecular underpinnings of C15 biology and immunomodulation to facilitate greater understanding of its molecular mechanisms and potential targeting as an anti-OPXV therapeutic strategy. This work will be completed at CHOP under the guidance of Drs. Laurence Eisenlohr and Nikolaos Sgourakis. Aim 1 will establish the functional necessity and outcome of C15 proteolytic processing using i) mutagenesis of potential cleavage sites coupled with T and NK cell functional assays and ii) systematic deletion coupled with analysis of proteolytic processing and sub-cellular localization. Aim 2 will define the C15 structural domains that are necessary for its antagonism of both NK and T cells. Here, we will use i) structure-guided deletions to identify the contributions of putative domains to immunomodulatory function and ii) recombinant protein technology to solve the structure and evaluate the activity of a putative MHC class I-like domain. Together, these aims will enable critical mechanistic insights into a potent virulence factor conserved across all pathogenic OPXVs, linking critical processing events and structural domains to virulence. Ultimately, this work will lay the foundation for further studies to investigate the targeting of B22 family proteins as a potential antiviral strategy against OPXVs. Furthermore, this work will provide critical training in techniques, data analysis and scientific communication that will support a career as an academic principal investigator.