Discovery of a [4Fe-4S] cluster in the PRRSV Nsp1α leader protease reveals host-virus interplay in its downstream functions

Porcine reproductive and respiratory syndrome virus (PRRSV; <i>Betaarterivirus suid</i>) is a major global threat to swine production, yet effective antiviral therapies are lacking. The leader protease Nsp1α is essential for viral replication and innate immune suppression, and its N-terminal zinc-finger (ZF) domain is critical for function, although its molecular role remains unclear. Here, we show that the ZF domain plays only a minor role in protease activity and that Nsp1α is largely inactive following release from the polyprotein. Using Mössbauer and UV/visible spectroscopy combined with chemoproteomics, we demonstrate that the ZF site binds not only Zn but also a [4Fe-4S] cluster. Notably, the Fe-S cluster, but not Zn, allosterically modulates residual protease activity. Nsp1α directly engages the cytosolic iron-sulfur cluster assembly machinery via CIAO1 and competes with the Fe-S carrier CIAO3, establishing the [4Fe-4S] cluster as a <i>bona fide</i> cofactor. These findings redefine Nsp1α as an Fe-S-dependent viral protein and reveal new opportunities for metal-targeted antiviral strategies.