Location: Virus and Prion Research
Project Number: 5030-32000-118-008-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2015
End Date: Dec 31, 2019
Objective:
Porcine reproductive and respiratory syndrome (PRRS) is the most important disease economically in the U.S. swine industry. Caused by a virus, PRRS virus (PRRSV)-infected pigs typically display a delayed onset of neutralizing antibody production and a weak cell-mediated immune response. PRRSV has been shown to inhibit synthesis of type I interferons (IFNs) and thereby suppress innate immunity. A viral ovarian tumor domain protease (vOTU) has been identified in PRRSV nonstructural protein 2 (nsp2), linked to immune suppression by its ability to reverse modification of host proteins by ubiquitin (Ub) and Ub-like proteins, in addition to its role in viral replication. As certain ubiquitination events are known to play a role in innate immune signaling, PRRSV vOTUs have been suggested as a possible virulence factor. Understanding the role of the vOTU in pathogenesis had been previously limited by a lack of biochemical information on vOTU activity as well as the necessity for animal experiments using recombinant viruses to discern true phenotypic differences in pathogenicity. We recently provided the first biochemical insight into PRRSV vOTU activity and specificity, Deaton et al, (2014), demonstrating divergence of vOTU substrate specificity between viral strains, a key finding which now requires the additional research in order to establish role of the vOTU in PRRSV pathogenicity.
We hypothesize that PRRSV vOTUs from highly pathogenic, moderate, and low virulence strains vary in activity and substrate specificities. Further, we hypothesize that these differences in activity and substrate specificity correspond to the ability of these viruses to evade the host immune system and thus play a critical role in disease severity.
Specific Objectives
1. Cooperator will examine effects of viral ovarian tumor domain protease (vOTU) sequence diversity on deubiquitinating and deISGylating activities.
2. ARS will aid in mutant virus production, animal studies and subsequent analysis to define the contribution of vOTU activity in subverting the innate immune response.
Approach:
Objective 1 will be completed using chemically-defined substrates and established biochemical assays to reveal the effects of viral ovarian tumor domain protease (vOTU) sequence diversity on the ability of divergent porcine reproductive and respiratory syndrome virus (PRRSV) strains to recognize and cleave mono-ubiquitin (Ub), poly-Ub and ISG15 in order to develop a predictive model for vOTU activity for newly emerging, uncharacterized PRRSV strains. This work will be facilitated by structural studies of multiple vOTUs and correlation with biochemical data. Objective 2 will focus on engineering and examining PRRSVs that possess inactive vOTUs or vOTUs with shifted substrate specificity. Swine will then be inoculated with selected engineered PRRSV strains to examine the effects of deubiquitinating and/or deISGylating activities on pathogenicity, as indicated by viral replication/fitness, suppression of interferon(IFN)-alpha/beta, inflammatory cytokine activity, and disease progression/outcome.
