Nidoviruses are an order of positive-sense RNA viruses, which include the families Arteriviridae and Coronaviridae. Nidoviruses express their complement of non-structural proteins (nsps) as a single polyprotein, which is cleaved into functional domains by proteases encoded within. The equine arterivirus (EAV) and Middle East respiratory syndrome coronavirus (MERS-CoV) encode for papain-like protease domains within nsp2 and nsp3, respectively, which serve a replicative role through their polyprotein processing activities. During infection, the host innate immune response is triggered by incoming pathogens, and engages multiple signalling pathways which are in-part dependent on the post-translational modification by ubiquitin (Ub). These signalling processes are tightly regulated by deubiquitinases (DUBs), which remove Ub from their cellular targets in order to reverse their effects. The EAV papain-like protease 2 (PLP2) and the MERS-CoV papain-like protease (PLpro), in addition to their replicative functions, were proposed to interfere with the induction of the cellular innate immune response to infection by acting as deubiquitinating enzymes. The fact that these enzymes rely on a single active site in order to carry out both DUB and polyprotein processing activities complicates our ability to assess the role of these functions independently. Here, the crystal structures of EAV PLP2, and the MERS-CoV PLpro in covalent complex with their Ub substrates were determined, and structure-guided mutagenesis was used to selectively remove DUB activity permitting the independent study of their DUB activities. Specific mutations targeting the Ub-binding interface of these enzymes inhibited DUB activity while permitting replicative polyprotein processing. Studies using these DUB-deficient enzymes demonstrated directly their role in the down regulation of cellular innate immune responses. In an effort to further define the molecular basis for substrate recognition by these viral DUBs, their structures were determined in complex with the antiviral Ub-like molecule ISG15, permitting further structural characterization of substrate recognition. In addition to the structural characterization of PLpro in complex with cellular substrates, the structures of PLpro in complex with novel, selective Ub-based inhibitors of the enzyme were determined. These inhibitors bound with high affinity mediated by a combination of hydrophobic and hydrogen-bonding interactions, inhibiting polyprotein cleavage, innate immune suppression and viral replication in cell culture.