Figure 7. Effect of inhibitors on RVFV replication. HSAECS were plated at 56104 cells per well in a 96-well plate and pretreated with either DMSO or indicated inhibitors for 5 h prior to RVFV infection at MOI of 0.002 as described in Materials and Methods. Following infection, fresh medium containing inhibitors was added. Supernatants were collected at 48 h.p.i., viral RNA extracted and analyzed by q-RT- PCR. * p = 0.0002, ** p = 0.031 (t-test) relative to mock control. doi:10.1371/journal.pone.0013805.g007 progressive accumulation of cells in the sub-G1 phase of cell cycle characteristic of cell death during the course of infection. HSAECs pretreated with the inhibitors for 5 h were infected with RVFV MP-12 at MOI 0.002. Supernatants were collected at 48 h.p.i. and analyzed by q-RT-PCR to determine the influence of these inhibitors on viral replication. We found that the cells treated with SB203580 demonstrated increased viral release (p = 0.0002) compared to the untreated cells (Fig. 7). The inhibitor of JNK did not show a statistically reliable change, indicating that JNK pathway might not control viral replication. However, the MEK1/ 2 inhibitor selectively affecting phosphorylation of the downstream ERK1/2 decreased viral titer (p = 0.031). No cell toxicity was observed at the concentrations of inhibitors used, indicating that the decrease in viral replication was not due to cell death (data not shown). These results suggested that p38 activity represents the protective cell response while activation of ERK1/2 pathway contributes to viral replication. Mechanisms of these effects are a subject of ongoing investigation. Discussion Host cell signaling is considered as one of the targets for future antiviral therapies. However, very little information is currently available regarding the RVFV infection in susceptible cells relevant to inhalation exposure. We chose experimental conditions with low MOI, likely reflecting the low-dose exposure to the virus in the natural environment, and found the lung epithelial cells to be highly susceptible. The cytopathic changes in the cells exposed to virulent strain ZH-501 took place over the period of 72 h, which allowed us to monitor a gradual development of infection, with the focus to identify the major pathways involved in the regulation of cell death. In our study for the first time we undertook quantitative analysis of phosphoprotein signaling induced by RVFV in HSAECs using RPMA. This approach allowed us to analyze at the same time the changes in the levels of phospho-modifications, activated forms, or total levels for 60 proteins. The signaling pathways altered in HSAECs infected with RVFV are summarized in Fig. 8. Significant changes in PI3K-AKT, MAPK, apoptotic and p53 pathways were observed. The AKT pathway is known to be one of RVFV Cell Signaling the prominent anti-apoptotic programs in mammalian cells that leads to inactivation of numerous apoptotic stimuli [27–29]. PI3K- Akt pathway is an important mechanism through which viral infection influences various cell functions. Activating PI3K-AKT signaling can slow down apoptosis and prolong viral replication in both acute and persistent infection [reviewed in ref. 34]. Accumulating evidence suggests that the activity of PI3K or AKT is critical for survival of several viruses. Adenovirus relies on PI3K-mediated organization of actin filament for active internal- ization. Non-segmented negative-sense RNA viruses require AKT to enhance synthesis of viral RNAs. On the other hand, PI3K- AKT signaling is associated with up-regulating interferon response. Higher PI3K-AKT activity might impede viral propa- gation due to activation of cellular defenses. Influenza A virus requires active PI3K for penetration despite the negative effects of inducing immune response. Unlike most viruses, it was reported that VP1 protein of foot-and-mouth disease virus inhibits AKT to promote cell death [reviewed in ref. 34] RVFV inhibition of AKT phosphorylation in HSAECs can promote cell death. Identifica- tion of virus protein(s) inducing this anti-survival signaling may provide clues to virus pathogenicity. The conserved inhibitor of apoptosis (IAP) family plays an important role in regulation of apoptosis through modulation of caspases [32,33]. IAPs can bind directly to caspases, leading to either their inactivation or degradation. The most potent caspase inhibitor of the IAP family is X-linked IAP, XIAP [33,35]. In the RVFV-infected HSAECs, AKT and XIAP downregulation as well as the caspase upregulation are progressively increased and therefore likely predispose cells to apoptosis. Caspases -3, -6 and -7 are effector caspases for both the intrinsic and extrinsic apoptotic pathways. To distinguish between these apoptotic pathways we analyzed proteins upstream of effector caspases. The cysteine aspartyl protease caspase-9 plays a central role in the mitochondrial or intrinsic apoptotic pathway that is engaged in response to many apoptotic stimuli. Caspase-9 is activated in a large multimeric complex, the apoptosome, which is formed with apoptotic peptidase activating factor 1 (Apaf1) in response to the release of cytochrome C from mitochondria [36]. Cleavage of caspase-9 on two sides Asp315 and Asp330 are necessary for there activation. Once activated, caspase-9 cleaves and activates the effector caspases 3 and 7 to bring about apoptosis. This pathway is tightly regulated at multiple steps, including apoptosome and cleaved caspase-9 formation. We detected no statistically significant changes in the level of cleaved caspase-9 between uninfected and RVFV-infected HSAECs. Therefore activation of caspases -3 and -7 most likely took place through the extrinsic pathway. Increased level of HSP27 is consistent with this conclusion [30]. In this respect, manipulation with caspases is not unique to RVFV. Influenza virus was reported to induce apoptosis by extrinsic death signaling through Fas/Fas-associated death domain-containing protein (FADD)/caspase-8 pathway [37]. The level of cellular caspase-3 activity correlated directly with the amount of progeny virus produced in the host cell line infected with influenza virus [38]. Similarly, the inhibition of caspases 3 and 9 with synthetic inhibitors, along with reducing apoptosis, reduced infective adenovirus titer in a dose-dependent manner [39]. p53 acts predominantly as a transcription factor, regulating the expression of more then 100 target genes to initiate apoptosis, cell cycle arrest, DNA repair, cellular senescence as well as differentiation [40]. RVFV induced the increase in phosphoryla- tion of p53 on both apoptosis-related sides, on S15 and S46. However, our data do not show increased levels of apoptotic proteins transcriptionally regulated by p53 (Bax, Bad, Bim, Puma, and Bid). This indicates that RVFV might possess other PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e13805