Structure-Function Analysis of Mxra8 Interaction with Alphaviruses

SUMMARY Alphaviruses are among the most important disease-causing, arthropod-borne viruses for humans. This genus includes viruses (e.g., chikungunya (CHIKV), Mayaro (MAYV), O’nyong’nyong (ONNV), and Ross River (RRV) viruses) that cause debilitating acute and chronic polyarthritis, have affected millions of people globally, and are emerging and re-emerging threats. Despite their epidemic potential, there are no specific therapies for alphaviruses. We previously used a CRISPR/Cas9-based screen to identify MXRA8 as a novel receptor for multiple arthritogenic alphaviruses including CHIKV, RRV, MAYV, and ONNV, a finding reproduced by several other laboratories. The identification of a receptor enabled us to generate soluble MXRA8- Fc decoy proteins that mitigate infection and disease in mice caused by multiple arthritogenic alphaviruses. In this renewal application, our collaborative team will continue to study the fundamental role of MXRA8 in alphavirus entry and pathogenesis. We will use cryo-electron microscopy to obtain a series of high resolution (< 3.2 Å) reconstructions of MXRA8 bound to multiple arthritogenic alphaviruses and use this information along with directed evolution to design MXRA8 decoy molecules that bind with greater affinity and neutralize infection more potently. We also will perform infection studies in conditional knockout mice and non-human primates (NHPs) with recombinant CHIKV strains that can or cannot bind to MXRA8 to further define its role in pathogenesis of acute and persistent disease. Finally, we will validate and perform structure- function analysis on new candidate MXRA8-independent entry factors for arthritogenic alphaviruses that are being identified through novel CRISPR-Cas9 gain-of-function and loss-of- function genetic screens. The innovative experiments in this renewal application will define fundamental aspects of alphavirus biology that enhance our understanding of how infection, tissue targeting, and disease occurs. This information also may facilitate the development of countermeasures that disrupt MXRA8 interaction with alphavirus envelope proteins, which could form the basis of therapeutics that ameliorate disease of multiple alphaviruses.