Molecular Mechanisms of Host Responses to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically devastating pathogen affecting the global swine industry. Since the emergence of the virus in the late 1980s, vaccination strategies aimed to control the virus have not been very effective. Current commercial vaccines are generally protective against homologous or closely-related strains but ineffective at conferring heterologous protection against genetically-diverse strains of the virus. Consequently, emergence of variant and sometime more pathogenic strains of PRRSV continues in global swine herds. As such, there is a need for better understanding of the molecular mechanisms involved in the replication of the virus.

In order to better understand the molecular mechanisms of host responses to PRRSV replication, we first sought to evaluate the ability of the virus to induce stress granules (SGs) during PRRSV infection. SGs are intracellular, cytoplasmic aggregates of RNA-binding proteins (RBPs) and mRNA. Formation of SGs is observed upon cellular stress and ultimately function to arrest cellular translation to promote cellular survival until the stress has been remedied. Indeed, several viruses have been shown to modulate the SG pathways to facilitate viral replication and even suppress the host's immune response. However, it is currently unknown whether PRRSV modulates the SG response. First, we used confocal microscopy and fluorescent in situ hybridization (FISH) to determine the distribution of known SG marker proteins and cellular mRNAs. Our findings revealed that PRRSV induces a potent SG response at late time points post-infection, and that SGs were closely associated with viral replication complexes (VRCs). Subsequently, we demonstrated that SGs are dispensable for viral replication, as short hairpin RNA (shRNA)-mediated knockdown of critical SG components (G3BP1 and G3BP2) did not affect viral replication. Interestingly, we found that the PRRSV-induced SGs are formed in a PERK-dependent manner. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Further investigation into the PERK signaling pathway revealed that PRRSV induces a significant amount of ER stress upon the cell during viral infection, and that exogenous stress significantly impaired the ability of the virus to replicate in MARC145 cells. We also showed that PRRSV potently induces all three signaling branches of the UPR, including PERK. While PERK knockdown had no effect on cell viability or viral replication, it significantly upregulated the mRNA expression of interferon-β and interferon stimulated genes (ISGs). The results from our studies suggest a critical role for PERK in regulating the host innate immune response to PRRSV infection. Only with a better understanding of the underlying molecular mechanisms of PRRSV replication will we be able to rationally design more effective vaccines against the virus.