The roles of papain-like protease-related proteins in viral replication and host immunity

Abstract

Viral papain-like proteases (PLPs)-related proteins have been shown to be actively involved in host innate immunity manipulation and virus replication. In this dissertation, the research were focused on the elucidation of biological roles of nidoviral PLPs-related proteins in innate immunity suppression and viral RNA transcription regulation. Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) is the most prominent swine diseases worldwide. Understanding PRRSV pathogenesis and development efficient vaccines are highly required in swine industry. PRRSV nsp2-related proteins including nsp2 and two ribosomal frameshifting products-nsp2TF and nsp2N share the same N-terminal PLP2 domain. In chapter 2, nsp2TF and nsp2N were demonstrated to be critical for host innate immunity suppression at least through the PLP2 domain-mediated deubiquitination and deISGylation effects. The infection of nsp2TF/nsp2N knockout mutants significantly upregulated antiviral innate immune responses in vitro. Furthermore, manipulating the expression of nsp2TF/nsp2N could enhance innate and adaptive immunity in pigs, providing potential basis for modified live vaccine development. In addition to the PLP2 domain of PRRSV nsp2-related proteins, the biological roles and biochemical nature of the poorly investigated long mysterious PLP2 downstream region was also characterized in Chapter 3. This long unknown region is also shared by nsp2-related proteins. At first, the hyper-phosphorylation nature of nsp2-related proteins was demonstrated. Physical features of this uncharacterized region was then delineated, including two intrinsically disordered hypervariable regions spaced by a structured inter-species conserved domain. One critical phosphorylated residues in the conserved domain were later proved to be of great importance in recombinant virus rescue and subgenomic RNAs accumulation. Collectively, our investigations underline the pleiotropic effects exerted by nsp2-related proteins in virus life cycle and potential contributions with pathogenesis. In Chapter 4, potential functions of PLP encoded by other nidovirus was also investigated. We discovered a unique cross-order recombination event, in which the chimeric picornavirus-enterovirus G expresses the PLP gene, homologous to torovirus (ToV) PLPs. Like other nidoviral PLPs, the recombinant ToV-PLP was proved to be a highly active deubiquinase/deISGylase and potent innate immune antagonist. After PLP knockout, viral fitness is significantly decreased and the suppression on host ubiquitination/ISGylation is largely reduced. Furthermore, host antiviral innate immune responses have been greatly upregulated post PLP knockout mutant infection. Our study underscores potential contributions of PLP domain in viral pathogenicity, and further provides an ideal example for how recombination shapes virus evolution. In summary, studies in this dissertation highlight the critical roles of nidoviral PLPs-related proteins in host immunity manipulation and virus replication, and more importantly, potential links with viral pathogenicity and application in vaccine development.