Reverse-Phase Phosphoproteome Analysis of Signaling Pathways Induced by Rift Valley Fever Virus in Human Small Airway Epithelial Cells Taissia G. Popova1, Michael J. Turell2, Virginia Espina3, Kylene Kehn-Hall1, Jessica Kidd 1 , Aarthi Narayanan1, Lance Liotta3, Emanuel F. Petricoin III3, Fatah Kashanchi1, Charles Bailey1, Serguei G. Popov1* 1 National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia, United States of America, 2 Virology Division, United States Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America, 3 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia, United States of America Abstract Rift valley fever virus (RVFV) infection is an emerging zoonotic disease endemic in many countries of sub-Saharan Africa and in Egypt. In this study we show that human small airway epithelial cells are highly susceptible to RVFV virulent strain ZH-501 and the attenuated strain MP-12. We used the reverse-phase protein arrays technology to identify phosphoprotein signaling pathways modulated during infection of cultured airway epithelium. ZH-501 infection induced activation of MAP kinases (p38, JNK and ERK) and downstream transcriptional factors [STAT1 (Y701), ATF2 (T69/71), MSK1 (S360) and CREB (S133)]. NF- kB phosphorylation was also increased. Activation of p53 (S15, S46) correlated with the increased levels of cleaved effector caspase-3, -6 and -7, indicating activation of the extrinsic apoptotic pathway. RVFV infection downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473), along with phosphorylation of FOX 01/03 (T24/31) which controls cell cycle arrest downstream from AKT. Consistent with this, the level of apoptosis inhibitor XIAP was decreased. However, the intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by an increased level of the inhibitor of apoptosome formation, HSP27. Concentration of the autophagy marker, LC3B, which often accompanies the pro-survival signaling, was decreased. Cumulatively, our analysis of RVFV infection in lung epithelium indicated a viral strategy directed toward the control of cell apoptosis through a number of transcriptional factors. Analyses of MP-12 titers in challenged cells in the presence of MAPK inhibitors indicated that activation of p38 represents a protective cell response while ERK activation controls viral replication. Citation: Popova TG, Turell MJ, Espina V, Kehn-Hall K, Kidd J, et al. (2010) Reverse-Phase Phosphoproteome Analysis of Signaling Pathways Induced by Rift Valley Fever Virus in Human Small Airway Epithelial Cells. PLoS ONE 5(11): e13805. doi:10.1371/journal.pone.0013805 Editor: Man-Seong Park, Hallym University, Republic of Korea Received June 14, 2010; Accepted October 7, 2010; Published November 3, 2010 Copyright: 2010 Popova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Department of Energy grant DE-FC52- FC04NA25455. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: spopov@gmu.edu Introduction Rift Valley fever virus (RVFV) is a highly-pathogenic arthro- pod-borne Phlebovirus of the Bunyaviridae family that infects a wide range of vertebrate hosts. In humans RVFV infection can lead to encephalitis, retinitis, or fatal hepatitis associated with hemor- rhagic fevers. In ruminants it is associated with high mortality rates, abortion, and fetal deformities [1,2]. RVFV infection is an emerging zoonotic disease endemic in many countries of sub- Saharan Africa and in Egypt. During the last decade the number of devastating outbreaks increased progressively [3,4]. Although the major route of RVFV entry into the host is through mosquito bites, some evidence indicates that aerosol route of infection can be highly effective. Laboratory workers have acquired RVFV supposedly after inhalation of infectious aerosols generated by careless handling of infected tissues [5]. Also, several animal species have been shown experimentally to be highly susceptible to airborne RVFV [5,6]. From this point of view, RVFV is considered as a potential biothreat. However, the frequency of airborne RVFV transmission under natural conditions remains unknown and interaction of the virus with the potential cell targets in the respiratory tract has not been studied. The information on the molecular biology of RVFV and its interaction with host cells is limited. RVFV has a tripartite single- stranded RNA genome consisting of large (L), medium (M), and small (S) segments [7]. The L and M segments are of negative polarity and express, respectively, the RNA-dependent RNA polymerase L and the precursors to the glycoproteins Gn and Gc. Gn and Gc cleavage also generates a nonstructural protein (NSm) that has been recently identified as a suppressor of virus-induced apoptosis [8]. The S segment utilizes an ambisense strategy and encodes the nonstructural protein NSs in genome orientation and the nucleoprotein N in antigenome orientation. RVFV NSs protein is not essential for virus replication in cell culture [9], while it works as a major viral virulence factor in infected animals [10]. NSs is expressed early in virus infection prior to viral RNA replication. It inhibits the induction of interferon beta [11], suppresses the host innate immune system by downregulation of PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e13805