RVFV Cell Signaling Figure 6. RPMA data for caspase-3, -6, -7, and -9 and actin. X axis labels, time post infection (h.p.i); Y axis labels, protein level relative to untreated cells (%) at 0 h.p.i. Open triangles, closed triangles and open squares correspond to the low MOI, high MOI and uninfected cells, respectively. Data represent mean values and confidence intervals of two-tail t-test (a = 0.05, n = 3). doi:10.1371/journal.pone.0013805.g006 substrates [26]. We found that the phosphorylations by RVFV of STAT1 (Y701), ATF2 (T69/71), MSK (S360), CREB (S133), NF- kB (S536), and p53 (S15/46) infection were transiently increased in general agreement with activation of MAPKs’ phosphorylation (Table 1 and Fig. 4, 5). Downregulation of pro-survival signaling through decreased phosphorylation of pro-survival AKT and anti- apoptotic XIAP The PI3K-AKT network activated by cytokines or growth factors mediates intracellular signals to regulate a variety of cellular responses, including anti-apoptosis, proliferation, cell cycling, protein synthesis, glucose metabolism and telomere activity [27–29]. RVFV infection decreased the phosphorylation of AKT on its major activation site S473 without considerable changes in the total amount of AKT in comparison with uninfected cells (Table 1 and Fig. 4). In agreement with this, the inhibitor of apoptosis, XIAP100 [30–33], was decreased in the infected cells, further contributing to apoptosis. Our data also show that RVFV infection downregulated the level of LC3B which is involved in cell autophagy. Effect of p38, JNK and MEK1/2 inhibitors on RVFV replication Our results indicated that MAPK pathways regulating cell fate in response to different stimuli were activated during RVFV infection. Therefore, we were interested in determining if inhibitors of these pathways could affect RVFV replication. For this purpose, we chose to test the inhibitors of p38 (SB203580), JNK (JNK Inhibitor II) and ERK1/2 (MEK1/2 Inhibitor III) in cells infected with RVFV MP-12. In preliminary experiments we showed that the MP-12 strain induced ERK1/2, p38, and JNK phosphorylation in a manner similar to ZH-501 (Fig. S1). We also found that in agreement with the ZH-501 data, the MP-12- infected cells demonstrated the onset of apoptosis evident in the increased degradation of cellular DNA. Figure S2 shows PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e13805