Abstract
Viruses have existed alongside cellular life for virtually the entirety of the earth’s biological history. All viruses encode proteins that interface with host cellular pathways and facilitate replication of the viral genome. This thesis focuses on viral proteases that contain tetracysteine metal-binding motifs that are annotated as zinc-finger (ZF) domains. The implication of zinc (Zn2+) at these metal-binding sites is based on primary sequence annotation and crystallographic studies, approaches that are blind to oxygen sensitive metallocofactors such as iron-sulfur (Fe-S) clusters. Fe-S clusters share compatible ligation chemistry and tetrahedral coordination geometry with Zn2+, and the two cofactors can often occupy the same tetracysteine binding sites interchangeably, leading to the misannotation of Fe-S proteins as Zn-binding. These findings reveal an unrecognized intersection between Fe-S cluster biology and viral replication machinery, with direct implications for the development of novel antiviral therapeutics targeting metallocofactor coordination. Chapter one of this thesis outlines major techniques for studying and characterizing Fe-S clusters, specifically focusing on three major forms of spectroscopy (UV/vis, Electron paramagnetic resonance, and Mössbauer spectroscopies) that can distinguish between structural forms and redox states of these metalloclusters. Complementary approaches including chemoproteomics, nuclear magnetic resonance, and pulse EPR are also described.
Chapter two is focused on the leader protease Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) whose N-terminal ZF domain is shown to coordinate a [4Fe-4S] cluster rather than Zn2+. The Fe-S cluster, but not Zn2+ , allosterically modulates protease activity, and Nsp1α directly engages the host Fe-S cluster biosynthesis machinery through CIAO1, establishing the [4Fe-4S] cluster as a bona fide cofactor. These findings redefine Nsp1α as an Fe-S protein, further supporting the emerging paradigm of Fe-S cluster utilization on viral proteins.
Chapter three presents unpublished work on the papain-like protease (PLpro) of SARS-CoV, another ZF-containing viral protease whose annotated Zn2+ binding site is shown to coordinate an Fe-S cluster. Beyond metallocofactor identity, PLpro is demonstrated to interact with components of both the mitochondrial ISC and cytoplasmic CIA Fe-S assembly pathways, and its deubiquitinase activity is allosterically activated by the WD40-repeat (WDR) proteins CIAO1 and WDR20, with CIAO1 conferring substantially greater stimulation preferentially enhancing activity against ubiquitin conjugated substrates. Given that PLpro targets multiple non-redundant nodes of the innate immune signaling cascade, the selective potentiation of deubiquitinase activity may amplify its immunosuppressive function, revealing a novel mode of PLpro regulation with implications for viral pathogenesis and antiviral drug development.