RVFV Cell Signaling Figure 1. Quantitative analysis of the newly-formed virus released from HSAECs into culture supernatants. A. Plaque assay data are presented as lg (PFU/ml) in supernatants of RVFV-infected HSAECs for MOI of 0.002. B, C. Western blot of lysed cells using antibodies against peptide from the middle of RVFV M segment and against actin (B, for MOI of 0.002 and C, for MOI of 0.0002). The amount of cells loaded was 1.46104 cells per well. Band intensities (arbitrary units) of viral proteins above the background detected in uninfected cells are shown as graphs below of Western data. The positions of Gc, Gn and NSm proteins translated from the M-segment are shown with arrows. doi:10.1371/journal.pone.0013805.g001 determined by staining with Sypro Ruby Protein Blot Stain of four randomly selected slides throughout the print run (Fig. 3). Similar levels of total protein were detected in infected and uninfected cells at the same time points post infection, likely reflecting cell growth without a detectable lysis up to 48 h.p.i. However, due to the progressive cytopathic effect of the virus, cell lysis became evident at 72 h.p.i. Since the RPMA analysis reflects the levels of intracellular proteins, all data were normalized to the total level of cellular proteins. The normalized values allowed direct comparison of results during the time course of infection. Of the 60 tested signaling proteins, the relative levels of 27 phosphorylated proteins and activated protein forms (such as cleaved caspases) showed statistically significant (p,0.05) differ- ences compared to uninfected cells at 48 and 72 h.p.i. (Table 1). Earlier time points did not demonstrate statistically significant differences between uninfected and RVFV-infected HSAECs. During the next 24 h the concentration of released virus increased more then 100-fold and was accompanied by a strong amplifica- tion of the phosphoprotein response. In order to validate the RPMA data, the levels of eight selected proteins were analyzed by Western blots using antibodies against phosphorylated and total forms of proteins (Fig. 4). Average band intensities from two independent Western blot experiments normalized to the total amount of cellular protein demonstrated good agreement with the data obtained from three independent RPMA experiments (Fig. 5). All 27 signaling proteins can be divided into three groups according to similarities in their temporal phosphorylation patterns (Table 1). In the first group the phosphorylation of the mitogen-activated protein kinase ERK1/2 and transcription factor STAT1, which is activated through a p38 (mitogen-activated protein kinase) MAPK-dependent pathway in response to IFN-a and other cellular stresses, were constantly increased. The same pattern was demonstrated by the cleaved caspases-3, -6 and -7, which are major components of extrinsic and intrinsic apoptotic pathways. The second group includes 13 proteins comprising the transcription factors: p53 (S15 and S46) controlling cell cycle arrest, DNA repair or apoptosis; MSK-1 (S360) activated by ERK and p38; anti-apoptotic STAT3 (S727) activated through the MAPK or mTOR pathways; ATF2 (T69/71) activated by SAPK/ JNK and p38 MAPK; NF-kB p65 (S536) playing a pivotal role in inflammatory and immune responses; and CREB (S133) mediat- ing numerous cellular responses. Among Group II are kinases SAP/JNK (T183/Y185) and p38 (T180), which regulate various cellular activities such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis; MAPK kinase MEK1/2 (S217/221); the 90 kDa ribosomal S6 kinase, p90RSK (S380), which is activated via coordinated phosphorylation by MAPKs, phosphoinositide-3-OH kinase (PI3K) and autophosphorylation in response to many growth factors, polypeptide hormones and neurotransmitters; insulin receptor substrate IRS-1 (S612), which phosphorylation is necessary for activation of AKT; and HSP27, conferring cellular resistance to adverse environmental changes. For group II, protein phosphorylation demonstrated a transient pattern of initial increase followed by a decline detectable mostly at the MOI of 0.002. However, at lower MOI of 0.0002, no decline of phosphorylation took place for the majority of these 13 PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e13805