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Abstract

Viruses have a limited coding capacity and therefore rely on hijacking host cellular machinery to facilitate infection. To achieve this, RNA viruses encode proteases that are involved in both viral polyprotein processing and the cleavage of host proteins. These cleavage events modulate cellular processes and evade innate antiviral defenses to promote replication and infection. Coxsackievirus B3 (CVB3) is a positive-sense single-stranded RNA virus that, like other picornaviruses, encode the 2A and 3C proteases (3Cpro). Recent advances in proteomics, such as terminal amine isotopic labeling of substrates (TAILS), have enabled the identification of hundreds of novel host proteins cleaved under viral infection. Among the host proteins identified by TAILS as potential CVB3 3Cpro targets, the RelA-associated inhibitor (RAI) was previously confirmed to be cleaved during infection, though the functional effects of cleavage remain unknown. In this thesis, I screened several proteins for cleavage under CVB3 infection and followed up on characterizing RAI cleavage and its functional outcomes. I confirmed that RAI is targeted by CVB3 3Cpro at the TAILS-predicted target site Q168↓G169. Therefore, cleavage of RAI at these sites generates at least two novel truncated fragments. It has been shown that protein fragments generated by viral protease cleavage can acquire novel functions to facilitate virus infection. Here, I found that overexpression of the N-terminal fragment generated by cleavage at Q168↓G169 inhibits NF-κB to a greater extent than the full-length protein. Furthermore, overexpression of this fragment also enhanced viral yield in host cells without affecting viral RNA replication. Together, these findings reveal a novel mechanism by which CVB3 modulates host immune signaling through 3Cpro-mediated cleavage of RAI. Understanding how viral proteases manipulate host substrates provides critical insight into virus-host interactions and can inform the development of antiviral strategies targeting protease activity or their host protein substrates.