Genotype 2 Strains of Porcine Reproductive and Respiratory Syndrome Virus Dysregulate Alveolar Macrophage Cytokine Production via the Unfolded Protein Response

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) infects alveolar macrophages (AMΦ) causing dysregulated interferon (IFN)-α and tumor necrosis factor (TNF)-α production through a mechanism(s) yet to be resolved. Here, we show that AMΦ infected with PRRSV secreted a reduced quantity of IFN-α following the cell exposure to synthetic dsRNA. This reduction did not correlate with reduced IFNA1 gene transcription. Rather, it coincided with two events that occurred late during infection and were indicative of translational attenuation, specifically, the activation of eukaryotic translation initiation factor 2α (eIF2α), and the appearance of stress granules. Notably, the typical rapid production of TNF-α by AMΦ exposed to lipopolysaccharide (LPS) was suppressed or enhanced by PRRSV depending on when the LPS exposure occurred after virus infection. If exposure was delayed until 6 h post-infection (hpi) so that the development of the cytokine response coincided with the time in which phosphorylation of eIF2α by the stress sensor PERK (protein kinase RNA (PKR)-like ER kinase) occurred, inhibition of TNF-α production was observed. However, if LPS exposure occurred at 2 hpi, prior to a detectable onset of eIF2α phosphorylation, a synergistic response was observed due to the earlier NF-κB activation via the stress sensor IRE1α (inositol-requiring kinase 1α). These results suggest that the asynchronous actions of two branches of the unfolded protein response (UPR), namely IRE1α, and PERK, activated by ER stress resulting from the virus infection, are associated with enhancement or suppression of TNF-α production, respectively. The activation of AMΦ is controlled by its microenvironment to deter excessive pro-inflammatory cytokine responses to microbes that could impair lung function. However, viral pneumonias frequently become complicated by secondary bacterial infections triggering severe inflammation, lung dysfunction, and death. Although dysregulated cytokine production is considered an integral component of the exacerbated inflammatory response in viral-bacterial co-infections, the mechanism responsible for this event is unknown. Here, we show that PRRSV replication in porcine AMΦ triggers activation of the IRE1α branch of the UPR, which causes a synergistic TNF-α response to LPS exposure. Thus, the severe pneumonias typically observed in pigs afflicted with PRRSV-bacterial co-infections could result from dysregulated, overly robust TNF-α production to opportunistic pathogens that is not commensurate with the typical restrained reaction by uninfected AMΦ. This notion could help design therapies to mitigate the severity of viral and bacterial co-infections.

Molecular and Cellular Mechanisms for PRRSV Pathogenesis and Host Response to Infection

“…For PRRSV, two branches of UPR pathways, IRE1-XBP1 and PERK-eIF2α, are activated. The UPR induction by PRRSV contributes to apoptosis (Huo et al, 2013;Yuan et al, 2016) and deregulates cytokine production in J o u r n a l P r e -p r o o f PAMs (Chen et al, 2017b). Further studies reveal that GP2a targets GRP78 for degradation .…”

“…Moreover, the UPR does not inhibit but instead stimulates efficient replication of PRRSV . In response to PRRSV infection, induction of UPR has been found not only contributing to PRRSV-induced apoptosis in host cells (Huo et al, 2013), but also involving in the regulation of viral replication and dysregulation of cytokine production in PAMs (Chen et al, 2017b).…”

Porcine Reproductive and Respiratory Syndrome Virus Infection Induces both eIF2α Phosphorylation-Dependent and -Independent Host Translation Shutoff

“…PRRSV induces both eIF2␣ phosphorylation-dependent and -independent host translation shutoff. Previous studies have indicated that PRRSV increases the phosphorylation of eIF2␣ (23,24). It is well known that the phosphorylation of eIF2␣ causes translation to stall (40).…”

“…PRRSV infection modulates normal cellular processes such as autophagy (16,17), apoptosis (18,19), and host innate immune responses (20)(21)(22). It is well known that PRRSV induces the phosphorylation of eIF2␣ and the appearance of stress granules (23,24). PRRSV GP4 and GP5 alter the gene expression profiles of stably transfected host cell lines (25).…”

A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain of African swine fever virus

“…This cell line has previously been shown to support replication of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) to high levels and it was demonstrated that efficacy of vaccine strains produced in this cell line was maintained at a high level following passage [2,3]. The ZMAC-4 cell line was used to investigate modulation of host stress responses to PRRSV infection [4]. In this report we describe experiments which demonstrate that ASFV isolates replicate in the ZMAC-4 cell line to levels similar to primary porcine macrophages without an adaptation step to the cell line.…”