PKR through its degradation in proteasomes and inhibition of eIF2-alpha phosphorylation [12], along with the general inhibition of cellular transcription [13]. Phosphorylation plays a key role in regulating many signaling pathways. Cell fate decisions in response to extracellular agents, including pathogenic invaders, are commonly mediated by phosphorylation-regulated signaling cascades that transduce signals into stimulus-specific actions, such as changes in gene expression pattern. Generally, both proapoptotic and prosurvival pathways are activated during viral replication. The possibility to interfere with virus replication either by enhancing antiviral signaling or by inhibiting proviral signaling can open a new avenue to antiviral treatments and prophylaxes. For example, in the proof-of-concept studies the inhibitors of Raf/MEK/ERK mitogenic kinase cascade and the IKK/NF-kB module reduce influenza virus titer in the lungs of infected mice after local aerosol administration into the trachea [reviewed in 14]. However, in the case of RVFV this potential opportunity has not been previously explored because of the limited knowledge of the intracellular events during infection. We therefore characterized the phospho- protein signaling by RVFV in the human small airway lung epithelial cells (HSAECs) as an in vitro model relevant to aerosol exposure. Using a high-throughput reverse-phase phosphopro- teome analysis during the time course of infection of HSAECs with the virulent wild-type RVFV strain ZH-501 we identified changes in major prosurvival and proapoptotic pathways controlled by the virus. Our analyses suggest a number of phosphoproteins as potential points of pharmacological interventions. Materials and Methods Reagents The ZH-501 strain of RVFV was isolated in 1977 from the blood of a 10-year-old Egyptian girl, who had a fatal RVFV infection [15]. This strain was passed twice in fetal rhesus monkey lung cells and once in Vero (African green monkey kidney) cells before use in this study. The attenuated RVFV MP-12 strain was a kind gift from Dr. Sina Bavari, United States Army Medical Research Institute of Infectious Diseases. The RVFV MP-12 was propagated and titrated using Vero cells. Experiments with live ZH-501 virus were carried out in the BSL-3 containment lab at the Virology Division of the United States Army Medical Research Institute of Infectious Diseases, MD, USA. Experiments with the MP-12 strain were carried out in the BSL-2 environment at the Biomedical Research Laboratory, George Mason University, Manassas, VA. Cell culture reagents were from CellGro (Herndon, VA). Antibodies against total and phosphorylated forms of the following proteins used for reverse-phase protein microarrays (RPMA) and Western blots were from Cell Signaling Technology (Beverly, MA) and were used at the dilutions indicated: 1:2000 for ERK1/2 (T202/Y204), 1:200 for STAT1 (Y701), 1:50 for Caspase-3 cleaved (D175), 1:100 for Caspase-7 cleaved (D198), 1:50 for Caspase-6 cleaved (D162), 1:1000 for p53 (S46), 1:50 for SAPK/JNK (T183/ Y185), 1:1000 for p53 (S15), 1:200 for p90RSK (S380), 1:100 for HSP27, 1:50 for MSK1 (S360), 1:50 for p38 (T180/Y182), 1:500 for MEK1/2 (S217/221), 1:100 for STAT3 (S727), 1:100 for ATF- 2 (T69/71), 1:50 for NF-kappaB p65 (S536), 1:400 for CREB (S133), for IRS-1 (S612), 1:1000 for Puma, 1:250 for FOX 01/03a (T24/32), 1:250 for Src (Y527), 1:1000 for Bim, 1:100 for Akt (S473), 1:100 for XIAP, 1:100 for LC3B, 1:50 for Cyclin D1, 1:100 for FADD (S194), 1:250 for GSK-3 alpha/beta (S21/9), 1:500 for PKR (T451), 1:50 for Beclin-1, 1:100 for mTOR (S2481), 1:500 for PTEN (S380), 1:100 for total p38, 1:50 for p70S6 Kinase (S371), 1:50 for AMPKalpha1 (S485), 1:500 for Bax, 1:100 for Bad (S136), RVFV Cell Signaling 1:100 for Elk1 (S383), 1:1000 for Bid, 1:500 for total SAPK/JNK, 1:200 for c-Raf (S338), 1:100 for caspase-9 cleaved (D315); 1:100 for caspase-9 cleaved (D330), 1:500 for Atg 5, 1:50 for FAK (Y397), 1:500 for PP2A A Subunit, 1:100 for ASK1 (S83), 1:200 for eNOS (S1177), 1:50 for b-Raf (S445), 1:200 for HSP90 (E289), 1:100 for total AKT, 1:1000 for total ERK1/2, 1:100 for Bcl-2 (T56), 1:100 for p27(T187),1:100 for Src family (Y416), 1:200 for beta actin, 1:100 for total p53, 1:100 for Jak1 (Y1022/1023), 1:50 for HSP70, 1:1000 for total PKR. The secondary HRP-labeled goat anti-rabbit antibody was from Cell Signaling Technology (Beverly, MA) and was used at the dilution 1:10000 for Western blot. The antibody against the viral antigen was from ProSci, Inc. (Poway, CA). It was raised against a peptide corresponding to a fragment near the center of the protein precursor translated from the M segment. It therefore detects both the precursor and the glycoprotein G1. Challenge of HSAECs with RVFV HSAECs (Cambrex Inc., Walkersville, MD) from anonymous donor were grown according to the vendor’s protocol in Ham’s F12 medium supplemented with nonessential amino acids, pyruvate, b- mercaptoethanol and 10% fetal calf serum (FCS) at 37uC, 5% CO2. The cells were adapted to these culture conditions during four passages and then were used for the preparation of the frozen stock. For reverse phase protein (RPMA) microarray experiments, confluent HSAECs at 106 cells per well in 6-well plates were challenged with RVFV ZH-501 at the multiplicity of infection (MOI) of 0.0002 and 0.002. 100 ml of RVFV suspension in a diluent (10% heat-inactivated FBS in Medium 199 with Earle’s salts, NaHC03 and antibiotics) containing 103.3 and 102.3 PFU of RVFV (MOI of 0.002 and 0.0002, correspondingly) or diluent were added to each well. Cells were incubated for 1 h at 37uC in the CO2 incubator and then 3 ml of HSAECs medium were added to each well and the plates returned to the CO2 incubator. At 0, 5, 24, 30, 48, 72 h post virus infection (h.p.i.), supernatant was removed, the cells were mixed with 500 ml of the lysis buffer, and immediately boiled for 10 min. The lysis buffer consisted of 1:1 mixture of T- PER Reagent (Pierce, IL) and 26Tris-glycine SDS sample buffer (Novex, Invitrogen, CA) in the presence of 2.5% b -mercaptoeth- anol, and protease and phosphatase inhibitors (16 Halt cocktail, Pierce). Lysed samples were stored at 280uC. Three independent cell challenge experiments were performed for the RPMA and Western blot analyses. RPMA and Western blot analyses Approximately 30 nl of each sample (equivalent to the amount of material from 40 lysed cells) were arrayed onto nitrocellulose slides (Whatman, MA) by direct contact printing using a high-resolution 2470 arrayer (Aushon Biosystems, Billerica, MA). Samples were printed as duplicates of an eight-point two-fold dilution series. The linear range of each dilution curve was then used for quantitative data comparisons between samples. Slides were stored at 220uC prior to detection. To estimate the total protein amount in each spot, several randomly selected slides were stained with Sypro Ruby Protein Blot Stain (Molecular Probes, Eugene, OR) and visualized on a Fluorchem imaging system (Alpha Innotech, San Leandro, CA). Slides were stained on a Dako Autostainer with specific antibodies using a biotin-linked peroxidase-catalyzed signal ampli- fication (Dako, CSA kit). Specifically, the arrayed slides were placed into Re-Blot solution (Chemicon, Temecula, CA) for 15 min, washed two times for 5 min each in PBS, placed into I-Block solution (Applied Biosystems, Foster City, CA) in PBS with 0.1% Tween-20 for at least 2 h, and then immunostained using an automatic slide stainer (Autostainer, Dako Cytomation) and the manufacturer-supplied reagents. Briefly, the slides were incubated PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13805