<div>The papain-like protease (PLP) domain of nonstructural protein 3 (nsp3) of the coronavirus (CoV) genome promotes viral replication by processing the CoV polyprotein (protease) and also antagonize innate immune responses by deubiquitinating (DUB) and deISGylating (deISG) host substrates. Selectively removing the DUB/deISG activities of PLP while keeping the protease activity intact is a potential strategy for designing a live attenuated virus. However, it is unclear in the literature the precise mechanism by which PLPs support CoV evasion of the innate immune system. Deciphering the substrate specificity of PLPs for host ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) can therefore help in the design of PLP mutants that selectively lack one activity for evaluating the DUB and deISG mechanism in CoV pathogenesis and replication. </div><div> In this dissertation, we investigate the structure and function of the single PLP (PLpro) from beta-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which are dangerous viral pathogens that emerged from a zoonotic source to cause infectious disease in the human population. Additionally, we translate the knowledge gained to the equivalent PLP2 from alpha-CoV porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV), which cause fatal disease in suckling piglets on industrial pork farms and household cats, respectively. The primary objective of this work is to rationally design PLP mutants across beta- and alpha-CoVs to help attenuate CoV infection, as no antiviral or vaccine exist for human CoVs and the efficacy of PEDV vaccines are an ongoing research topic. </div><div><br></div><div>In Chapter 1, different human, animal, and the bat origin CoV strains are introduced. The CoV life-cycle and virion structure are outlined, along with the replicase complex for viral replication. The multidomain nsp3 from alpha- and beta-CoV genomes are also described with a focus on the PLP domain and its proposed cleavage sites of the viral polyprotein. The discovery of the first viral protease DUB and the multiple activities of PLPs are defined, which includes a proposed model of how DUB versus deISG activities may act in the innate immune response. This leads into the therapeutic potential of PLP for an antiviral or live attenuated vaccine, which is followed by the introduction of live attenuated vaccines and the reverse genetics system. Next, proof of concept studies on PLP2 mutants are described and the introduction is concluded by stating the ultimate goal for the design of PLP mutants.</div><div><br></div><div>In Chapter 2, we hypothesize that the flanking ubiquitin-like (Ubl2) domain of MERS-CoV PLpro is not required for its enzymatic function. We characterize the specific activity, kinetics, substrate specificity, and inhibition of the PLpro enzyme with and without the Ubl2 domain and reveal that the Ubl2 domain does not significantly alter PLpro function. We determine the structure of the core PLpro, smallest catalytic unit to 1.9 Å resolution and observed no structural changes compared to the wild-type. Additionally, we demonstrate that a purported MERS-CoV PLpro inhibitor is nonselective in non-reducing conditions and should not be pursed for therapeutic use. We show that the core PLpro enzyme i.e. without the Ubl2 domain is a stable and robust construct for crystallization and is also thermally stable based on thermal melting studies with utility for structure-based drug design. </div><div><br></div><div>In Chapter 3, we shed light on the specificity of SARS-CoV PLpro towards Ub versus ISG15 by characterizing the specific activity and kinetic parameters of SARS-CoV PLpro mutants. In addition, the structure of SARS-CoV PLpro in complex with the C-terminal domain of ISG15 is determined and compared with the Ub-bound structure. Based on the structure and kinetic results, the altered specificities of SARS-CoV PLpro mutants Arg167Glu, Met209Ala, and Gln233Glu are compared with the wild-type. Arg167Glu mutant exhibits DUB hyperactivity and is expected to adopt a more favorable interaction with the Arg42 of Ub. At the same time, ARG167GLU contains a shorter side-chain that hinders interaction with the unique Trp123 of ISG15 for deISG activity compared to the wild-type. These results aid in the development of SARS-CoV PLpro mutants that have directed shifts in substrate specificity for Ub versus ISG15. </div><div><br></div><div>In Chapter 4, the process and antiviral activity of ISGylation is reviewed and how viruses can modulate host-derived versus virus-derived machineries to counteract ISGylation for viral infection. MERS-CoV PLpro is cross-reactive for Ub, but less is known about its specificity towards ISG15. In this study, we determine the structure of MERS-CoV PLpro bound with ISG15 to 2.3 Å resolution and reveal a small hydrophobic pocket of ISG15 that consists of P130 and W123, which differs from Ub hydrophobic patch. We design and determine the kinetic parameters for 13 PLpro mutants and reveal that MERS-CoV PLpro only has a single ubiquitin recognition (SUb1) site. Kinetic studies show that removing the charge of the R1649 greatly enhances DUB/protease activity while mutating in an Arg near R42 of Ub or ISG15 hydrophobic region is detrimental to both DUB/deISG activities. Kinetic experiments and probe-reactivity assays showed that Val1691Arg, Val1691Lys, and His1652Arg mutants are drastically reduced DUB/deISG activities compared to the wild-type. Overall, MERS-CoV PLpro mutants with alter kinetic profiles will be useful for discovery tools and DUB/deISG deficient mutants are great candidates for removing host cell antagonism activity by PLpro for live attenuated vaccines.</div><div><br></div><div>In Chapter 5, the goal is to translate the knowledge gained in Chapters 2-4 on beta-CoVs PLpro and evaluate the substrate specificity of alpha-CoVs FIPV and PEDV PLP2 for mutagenesis experiments. First, we design and purify the core PLP2 enzymes for kinetics. PLP2s are efficient DUBs that prefer Ub to ISG15 in vitro, and this preference is conserved in beta-CoV MHV PLP2 as well as alpha-CoV NL63 PLP2. We determine the structure of alpha-CoV PEDV PLP2 to 1.95 Å resolution and reveal the unique Zn-finger coordinating Cys3-His arrangement of the alpha-CoV genus that differs from past beta-CoV PLP crystal structures. To determine residues of the SUb1 site, we generate a homology model of FIPV PLP2 and overlay our PLP2 structures with MERS-CoV PLpro bound with Ub. In addition, we create electrostatic surface maps across coronaviral PLP subfamilies to evaluate the charge distribution of the SUb1 for the rational design of several FIPV and PEDV PLP2 mutants. We evaluate the turnover of PLP mutants for FRET-based substrates and reveal that His101ArgFIPV and Asn101ArgPEDV are drastically reduced in Ub-AMC activity while their peptide activities are within 2-fold of the wild-type. These mutants show delayed reactivity for Ub probes and no longer cleave Ub-chains displaying isopeptide bonds compared to the wild-type. Results from this study reveal a hot spot in both alpha- and beta-CoVs that can be used to selectively remove DUB activity of PLPs for generating a DUB deficient PLP enzyme. </div><div><br></div><div>In this dissertation, we investigate the substrate specificity of PLPs across alpha- and beta-CoVs and develop a fingerprint for Ub and also shed light on ISG15 recognition. Specifically, hot spots were identified in the SUb1 site of different PLPs, which recognize R42 and hydrophobic Ile44 of Ub. Position 97-98 of PLPs can be used to remove DUB activity by substituting an Arg, but usually effect protease function. Substituting an Arg at position 101 and 136 of coronaviral PLPs serve as the best strategy to remove DUB function while not hindering active site functionality. The DUB/deISG deficient mutants described will be useful for inhibiting the ability of PLPs to function in the innate immune response. Ultimately, this work provides a guide for identifying attenuating mutants in existing CoVs for live attenuated vaccines and also a blueprint for engineering PLPs from new emerging CoVs. </div>

L’étude a pour premier objet de découvrir les caractères communs aux jeux antiques, au jeu de paume et à la pelote basque à partir d’une étude des textes écrits par des contemporains de ces jeux. Puis, l’observation des héritiers de la paume, dans leur histoire et leur actualité, permet des comparaisons pour cerner héritages et créations à l’origine de la pelote. Dresser une carte des identités de la pelote basque, occupe une grande partie de l’ouvrage, à partir de l’analyse des archives des villes du Pays basque et des articles de presse, principalement Le Courrier de Bayonne (1851 - 1899). Des rares premières archives des XVIe et XVIIe siècles et des témoignages contemporains du jeu décrit jusqu’en 1899, on tire les mutations, les règles et les conditions spacio-temporelles ou socio-culturelles de la pratique de la paume/pelote basque, donc son histoire sur cette période. L’étude des lieux de mémoire, tant les aires de jeu que la langue basque, la géographie d’implantation de la pelote en France et dans le monde, ou encore l’installation de la tradition des fêtes basques initiées par Antoine d’Abbadie, rend compte d’autres éléments identitaires et de la complexité toujours plus étendue de “ la ” pelote basque. Enfin les représentations des jeux de balle et de la pelote, dans leurs formes, littéraire, des arts visuels, journalistique, publicitaire, sont questionnées pour mieux cerner, d’une part les symboles à l’œuvre depuis l’antiquité et, d’autre part, le phénomène pelote basque dans sa personnalité et sa modernité. Les premières archives et l’histoire du XIXe siècle montrent une pratique mêlant joueurs des Pays basque de France et péninsulaire mais une évolution différente des aires et des conditions se fera dans les années 1880 et la pelote se différencie durablement. Devenue sport au début du XXe siècle et sport international, la pelote assume des pratiques plurielles, des influences venues des pays d’Amérique et ses nombreux paradoxes. / The first aim of this thesis is to determine the commonalities between games of Antiquity, real tennis, and Basque pelota, based on a review of documents written by authors coeval to these games. Then, the observation of the descendants of real tennis, in their history and their actuality, allows for comparisons to identify inheritances and creations at the origin of Basque pelota. A large part of the work is devoted to mapping out the identities of Basque pelota, based on the analysis of town records in the Basque Country and press articles, mostly from Le Courrier de Bayonne (1851 - 1899). From the rare first records from the 16th and 17th centuries and testimonies contemporaries of the game described until 1899, we draw the mutations, the rules, and the spatio-temporal or socio-cultural conditions of the practice of real tennis / Basque pelota, thus its history over this period. The study of sites of memory, such as sports fields, Basque language, the geography of establishment of Basque pelota in France and in the world, or even the establishment of the tradition of Basque festival initiated by Antoine d'Abbadie, provides an account of other identity elements and of the ever growing complexity of "the" Basque pelota. Finally, the representations of ball games and pelota, in their literary, visual arts, journalistic, and publicity form, are questioned to better pinpoint, on the one hand, the symbols used since antiquity, and, on the other hand, the phenomenon of Basque pelota in its personality and its modernity. The first records and the history of the 19th century exhibit a practice mixing players from the Basque Countries of France and of Spain but a contrasting evolution of the fields and conditions will emerge in the 1880s, and Basque pelota will lastingly be differentiated. Institutionalised in the early 20th century and made international, Basque pelota adopts plural practices, influences from the Americas, and their many paradoxes.