RVFV Cell Signaling Figure 8. RPMA data presented as a diagram of signaling events in HSAECs infected with RVFV. Among the most prominent responses was activation of MAP kinases (p38, JNK and ERK) and the downstream transcriptional factors [STAT1 (Y701), ATF2 (T69–71), MSK1 (S360), and CREB (S133)]. STAT3 and NF-kB phosphorylation were also increased. Activation of p53(S15, S46), which typically accompanies transcriptional upregulation of the apoptotic genes correlated with the increased levels of cleaved effector caspase-3, -6 and -7 indicating activation of the extrinsic apoptotic pathway. RVFV infection also downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473). Consistent with this, the level of apoptosis inhibitor XIAP100 was decreased. The intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by increased level of the inhibitor of apoptosome formation, HSP27. doi:10.1371/journal.pone.0013805.g008 mechanisms of apoptosis downregulation through the intrinsic pathway, such as the NSm-dependent one [8], in addition to the upregulation of HSP27 discussed above. Members of the MAPK family are involved in signal transduction of apoptosis as well as cell growth and differentiation. During RVFV ZH-501 infection in HSAECs the phosphorylations of MAPKs including p38, JNK and ERK were found to be upregulated. ERK1/2 plays a central role in cell proliferation and differentiation [41–43]. At both MOIs, constant progression of ERK1/2 activation took place in HSAECs infected with RVFV in agreement with the increased phosphorylation on S380 of the upstream ERK1/2-activating kinase p90RSK. Activation of p90RSK was also evident in the increased phosphorylation of the downstream CREB. This pattern was consistent with a pro- survival host response through ERK1/2, although it did not exclude the role of the ERK signaling cascade in promoting RVFV replication, as it has been shown for influenza virus [44]. The JNK pathway is activated by cellular stress and by cytokine induction. JNK pathway activity can mediate apoptosis, prolifer- ation, or survival, depending on the stimuli and cellular conditions. Sustained JNK activity is necessary for cellular homeostasis, whereas strong stress stimuli in non-transformed cells primarily lead to JNK-mediated apoptosis. The activated form of JNK phosphorylated on T183 and Y185 was increased in RVFV- infected HSAECs initially and then dramatic dephosphorylation was observed (more then 10-fold at 72 h.p.i. in comparison with 48 h.p.i) by the higher dose of infection, indicating that it is one of the major RVFV targets. PLoS ONE | www.plosone.org 11 November 2010 | Volume 5 | Issue 11 | e13805