While PRRSV has been recently shown
While PRRSV has been recently shown to induce SGs, the exact nature of SG formation and its underlying mechanism of regulating antiviral immune response during PRRSV infection remain unclear (Zhou et al., 2017). Formation of PRRSV-induced SGs was observed in MARC145 Ketorolac (Chen et al., 2018) and PAM cells (Zhou et al., 2017). However, previous work in PAM cells is very limited, as only a single SG marker (TIAR) was used. Since SGs are very diverse in composition and function, the structures reported by Chen et al cannot be concluded as indeed bona fide SGs without the use of additional markers and assays. Therefore, it is critically important to more extensively investigate the formation and underlying mechanism of PRRSV-induced SGs. The formation of SGs in response to PRRSV infection appears to be dependent on PERK-mediated eIF2a phosphorylation. The eIF2a pathway is a critical regulator of cellular translation in response to various stress conditions. PERK is one of 4 known eIF2a kinases that respond to cellular stress and is activated upon ER stress. Such stress can be triggered by an overload of unfolded proteins in the ER, as occurs during infection of most viruses. Treatment of cells with PERK inhibitor prevents eIF2a phosphorylation and subsequent SG formation in PRRSV infected cells (Zhou et al., 2017). PRRSV could potentially be using the eIF2a pathway to its advantage in order to facilitate viral replication. A reduction in global translation could result into a reduction in overall antiviral signaling, thus creating a favorable environment suitable for replication. Additional work is therefore warranted in the future to determine the true nature of the PRRSV-induced SGs and the pathways involved in their formation. Importantly, FISH analysis and confocal microscopy revealed that mRNA, a hallmark characteristic of functional bona fide SGs, is also recruited to the PRRSV-induced SGs. Considering the composition of the PRRSV-induced SGs as well as the fact that their disassembly is facilitated by CHX, we conclude that the PRRSV-induced SGs are bona fide SGs. Indeed, the finding that mRNA is recruited to the PRRSV-induced SGs suggests that they may be acting as a storage repository for RNA transcripts. While SGs have been shown to temporarily store mRNAs until resolution of stress conditions and granule disassembly, the exact fate of these transcripts is not fully understood (Panas et al., 2016). It is currently thought that RNA transcripts retained in SGs are released back into the cytoplasm for subsequent translation and protein synthesis. Alternatively, if stress is not resolved, the transcripts could be shuttled to P-bodies instead (Mollet et al., 2008; Brengues et al., 2005). In this study, we found that the PRRSV induced SGs are closely associated with P-bodies and VRCs. The SG marker G3BP1 and P-body marker Dcp1a were found to be in close proximity to the sites of PRRSV replication, as indicated by the presence of the viral protease, NSP2. These data suggest that mRNA is potentially stored and transferred between these sites during PRRSV infection. Moreover, we show that the rate of host cellular protein synthesis is drastically reduced during PRRSV infection. Importantly, we demonstrated that this reduction of protein synthesis is coordinated with SG formation during PRRSV infection. Taken together, these data suggest a possible mechanism in which the PRRSV-induced SGs function to sequester mRNAs and preclude them from being translated. Alternatively, this data also suggests that the PRRSV-induced SGs could also play an important role in regulating the PRRSV lifecycle and facilitate the switch from genome translation to replication. While other viruses have been shown to utilize SG components to enhance their replication and carry out infection, SGs seem to be dispensable for PRRSV replication. In this study, we used shRNAs to knock down both G3BP1 and G3BP2 individually and together and observed no difference in PRRSV replication as determined by plaque assay. However, additional parameters need to be investigated in the future before any definitive conclusions are made. Specifically, the role of SGs in disease pathogenesis warrants further investigation, as their function in regulating the expression of specific cytokines during PRRSV infection has yet to be fully elucidated. Interestingly, when G3BP1 and G3BP2 were knocked down individually, we observed a strong up-regulation of the other protein (Fig. 6A). When both proteins were knocked down together, we observed a weaker reduction in both proteins compared to individual knock-downs. One potential explanation for this observation is a form of genetic compensation: when one protein is reduced, the cell up-regulates expression of another related protein to compensate for the loss.