Activation of the p38 MAPK pathway is required for apoptosis induction in several different cellular models [20–23]. Addition- ally, stress-elicited p38 activation was shown to cause G2/M cell cycle arrest and to regulate the cell cycle through modulation of p53 and p73 tumor suppressor proteins [24] RVFV infection in HSAECs demonstrates similar patterns of phosphorylation for p38 and JNK for both of these pathways important for cell survival. Phosphorylated p38 proteins can activate an array of transcription factors, including ATF2, CHOP1, MEF2, p53, STAT1 and Elk1. For JNK a classical substrate is the transcription factor c-Jun along with ATF2, Elk1, MEF2c, p53, STAT1, and c-Myc. Our data for STAT1 (Y701), ATF2 (T69–71) and MSK1 (S360) indicate their coordinated upregulation with MAPKs and implicate RVFV in a widespread control of cellular transcription. Taking into account the importance of MAPK pathways for cell fate we tested the effect of p38, JNK and ERK1/2 phosphory- lation inhibitors on replication of RVFV MP-12 strain, which demonstrated activation of MAPKs similar to ZH-501. Using the inhibitor-pretreated cells, we found that viral growth can be promoted or inhibited by the p38 and ERK1/2 pathway inhibitors, correspondingly, while JNK cascade did not exert a strong effect on the amount of released virus. These data support a potential utility of phosphoprotein signaling cascades as host targets for treatment of viral infections. However, a strong influence of MOI on the level and dynamics of transient cell responses (Table 1) brings up an additional level of complexity, which needs to be addressed in future studies. For example, phosphorylation of several proteins from group II at certain time demonstrated either increasing or decreasing trends, depending on the progress of infection. We therefore suggest that potential signaling-based therapeutic interventions would need to take into account not only the nature but also a magnitude of the particular response for adequate treatment. Future experiments with specific inhibitors administered at different times post infection will help understand the significance of the identified pathways for survival/ References 1. Balkhy HH, Memish ZA (2003) Rift Valley fever: an uninvited zoonosis in the Arabian peninsula. Int J Antimicrob Agents 21: 153–157. 2. Peters CJ, Meegan JM (1989) Rift Valley fever. CRC Press: Boca Raton, FL. 3. Gerdes GH (2002) Rift valley fever. Vet Clin N Am Food Anim Pract 18: 549–555. 4. Flick R, Bouloy M (2005) Rift Valley virus. Curr Mol Med 5: 827–834. 5. Brown JL, Dominik JW, Morrissey RL (1981) Respiratory infectivity of a recently isolated Egyptian strain of Rift Valley fever virus. Infect Immun 33: 848–853. 6. Easterday BC (1965) Rift Valley fever. Adv Vet Sci 10: 65–127. 7. Elliott RM, Bouloy M, Calisher CH, Goldbach R, Moyer JT, et al. (2000) Family Bunyaviridae, p. 614–616. In Van Regenmortel MHV, Fauquet C, Bishop DHL, Cartens EB, Estes MK, et al. (ed.), Virus taxonomy: classification and nomenclature of viruses. Seventh report of the International Committee on Taxonomy of Viruses. Academic Press, Inc.: San Diego, CA. 8. Won S, Ikegami T, Peters CJ, Makino S (2007) NSm protein of Rift Valley fever virus suppresses virus-induced apoptosis. J Virol 81: 13335–13345. 9. Ikegami T, Won S, Peters CJ, Makino S (2006) Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene. J Virol 80: 2933–2940. 10. Bouloy M, Janzen C, Vialat P, Khun H, Pavlovic J, et al. (2001) Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs. J Virol 75: 1371–1377. 11. Le May N, Mansuroglu Z, Leger P, Josse T, Blot G, et al. (2008) A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells. PLoS Pathog 4: e13. 12. Ikegami T, Narayanan K, Won S, Kamitani W, Peter CJ, et al. (2009) Rift Valley fever virus NSs protein promotes posttranscriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation. PLoS Pathog 5: e1000287. 13. Le May N, Dubaele S, Proietti De Santis L, Billecocq A, Bouloy M, et al. (2004) TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus. Cell 116: 541–550. 14. Ludwig S (2009) Targeting cell signalling pathways to fight the flu: towards a paradigm change in anti-influenza therapy. J Antimicrob Chemoth 64: 1–4. RVFV Cell Signaling death cell decision in RVFV-infected HSAECs. The experiments with aerosol RVFV-infected animals are also forthcoming to analyze virus-induced signaling in the lung and to reconcile these findings with our in vitro model. Supporting Information Figure S1 Induction of ERK1/2, p38, and JNK by RVFV MP- 12. Western blots were performed on lysates of RVFV MP-12- infected HSAECs at indicated times post infection with MOI of 0.002 (for ERK1/2, p38 and JNK). Open and grey bars show band intensities of uninfected and infected cells, correspondingly, calculated relative to uninfected cells at 24 h.p.i. Found at: doi:10.1371/journal.pone.0013805.s001 (0.92 MB TIF) Figure S2 Sub-G1 cell cycle fraction of HSAECs is increased during RVFV infection. HSAECs were mock infected or infected with RVFV MP12 at MOI of 3.0. Cells were collected at 6, 24, and 48 h.p.i. and cell cycle analysis performed by propidium iodide staining. Flow cytometry data were acquired on a Becton Dickinson FACScaliber with a 488-nm argon laser. Acquisition and analysis were performed with CELLQuest software. A. The percent of the sub-G1 population relative to the total number of cells as average of two independent samples. Open bars, mock- infected cells; gray bars, RVFV-infected cells. B. Histogram of the sample at 48 h.p.i. illustrating the position of the sub-G1 fraction gate. Found at: doi:10.1371/journal.pone.0013805.s002 (0.71 MB TIF) Author Contributions Conceived and designed the experiments: TGP MJT LL EFP CB SP. Performed the experiments: TGP MJT VE KKH JK AN. Analyzed the data: TGP FK SP. Contributed reagents/materials/analysis tools: VE LL EFP. Wrote the paper: TGP KKH FK SP. 15. Meegan JM (1979) The Rift Valley fever epizootic in Egypt 1977-78. 1. Description of the epizootic and virological studies. Trans R Soc Trop Med Hyg 73: 618–623. 16. Wulfkuhle JD, Edmiston KH, Liotta LA, Petricoin EF, III (2006) Technology insight: pharmacoproteomics for cancer –promises of patient-tailored medicine using protein microarrays. Nat Clin Pract Oncol 3: 256–268. 17. Garrido C, Bruey JM, Fromentin A, Hammann A, Arrigo AP, et al. (1999) HSP 27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB J 13: 2061–2070. 18. Davis R (2000) Signal transduction by the JNK group of MAP kinases. Cell 103: 239–252. 19. Weston CR, Davis RJ (2007) The JNK signal transduction pathway. Curr Opin Cell Biol 19: 142–149. 20. Li S-P, Junttila MR, Han J, Ka¨ha¨ri V-M, Westermarck J (2003) p38 Mitogen- activated protein kinase pathway suppresses cell survival by inducing dephosphorylation of mitogen-activated protein/extracellular signal-regulated kinase 1,2. Cancer Res 63: 3473–3477. 21. Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, et al. (2001) ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2: 222–228. 22. Porras A, Zuluaga S, Black E, Valladares A, Alvarez AM, et al. (2004) P38 alpha mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli. Mol Biol Cell 15: 922–933. 23. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME (1995) Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326–1331. 24. Bulavin DV, Fornace AJ Jr (2004) p38 MAP kinase’s emerging role as a tumor suppressor. Adv Cancer Res 92: 95–118. 25. Gauthier ML, Pickering CR, Miller CJ, Fordyce CA, Chew KL, et al. (2005) p38 regulates cyclooxygenase-2 in human mammary epithelial cells and is activated in premalignant tissue. Cancer Res 65: 1792–1799. 26. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, et al. (2001) Mitogen activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22: 153–183. PLoS ONE | www.plosone.org 12 November 2010 | Volume 5 | Issue 11 | e13805