Investigations into the role of stress granule formation during respiratory syncytial virus infection
Lindquist, Michael Edward
Several viruses are known to induce stress granules (SGs), however, a role for these structures during viral infection remains unknown. We first showed that respiratory syncytial virus (RSV) induces SGs starting approximately 12 hours after infection in cultured cells. We then generated a cell line with limited capacity to form stress granules by knocking down expression of the SG assembly protein, G3BP. When G3BP knockdown cells were infected with RSV, we observed a significant decrease in viral titer in comparison to control cells. Since SGs are known to contain RNAs, we performed extensive studies with sensitive novel viral RNA probes in live cells to determine the location of production of viral RNA in infected cells. We showed that at the earliest time points in viral replication, viral RNAs appear in discrete granules containing viral proteins (termed inclusion bodies) that were distinct from SGs. These data suggested that inclusion bodies and not SGs were the primary site of replication of viral RNA. We then determined the mechanism by which RSV induces SGs. We showed that RSV infection activates PKR, which subsequently phosphorylates eIF2α and results in SG formation. In addition, when PKR expression was inhibited, we observed a decrease in viral-mediated SG formation, although we found no change in viral titer. In contrast, a PKR inhibitor 2-AP that also may affect other cellular kinases potently inhibited RSV replication. These data indicate that RSV specifically induces a stress response including activation of PKR and eIF2α with formation of host cell SGs, which appear to play an enhancing role in viral replication.