Structural and mechanistic studies of multifunctional proteases in virus replication and host immune evasion

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The synthesis and posttranslational cleavage of polyproteins is a common genome expression strategy used by positive-strand (+)RNA viruses. Cleavage of the polyproteins into functional units is essential to the virus and is carried out by virus-encoded proteases, many of which also target cellular proteins to disrupt host antiviral responses and to assist with virus replication. My NSERC research program explores the molecular basis underlying the multifunctional role of viral proteases in the replication and host immune evasion strategies of +RNA viruses. I currently focus on +RNA viruses from the order Nidovirales, which contains viruses of animal and human importance, including arteriviruses, such as equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV), as well as the severe acute respiratory syndrome (SARS) and the emerging Middle-East respiratory syndrome (MERS) coronaviruses. These viruses express two polyproteins that contain a number of non-structural proteins (nsps), a subset of which include active protease domains. PLP2 and PLpro are proteases located within nsp2 and nsp3 of arteriviruses and coronaviruses, respectively. Remarkably, in addition to acting as endopeptidases that cleave sites in the viral polyproteins, they also act as isopeptidases to remove the posttranslational modifier proteins ubiquitin (Ub) and ISG15 from cellular proteins to suppress Ub- and ISG15-dependent host antiviral pathways. In contrast, nsp1ß is a protease of PRRSV that also participates in polyprotein cleavage, yet its ancillary function is to assist virus replication by forming an RNA binding complex with the host protein PCBP2 to control programmed ribosomal frameshifting events that yield additional viral proteins during virus genome translation. Using molecular and structural biology methods, I aim to provide a detailed understanding of the diverse roles that PLP2, PLpro and nsp1ß play during the virus life cycle. I will identify the structural features that enable PLP2 and PLpro to disrupt Ub- and ISG15-dependent antiviral pathways, develop inhibitors of PLP2 and PLpro based on tight-binding Ub mutants to probe their substrate binding properties and catalytic mechanisms, and reveal how nsp1ß forms an RNA binding complex with PCBP2 to control ribosomal frameshifting. The research will yield important new insights into how viral proteases subvert host antiviral responses and direct the non-canonical translation of viral proteins, making it of wide interest to virologists, biochemists and cell biologists. Trainees engaged in the research will benefit from a highly productive research program in structural and molecular biology. They will gain the skills they need to become highly qualified personnel in academia or industry where a working knowledge of molecular and structural biology, biochemistry and biotechnology is required.