RVFV Cell Signaling Figure 2. Example of RPMA slides stained with antibodies against total and phosphorylated forms of SAP/JNK, ERK and AKT and against phosphorylated form of p-38 and PTEN. Portions of slides stained with antibodies against total and phosphorylated protein forms are shown for untreated (Control) and RVFV-infected HSAECs at 48 and 72 h.p.i. Low and high MOI correspond to 0.0002 and 0.002, respectively. Each sample along with eight 26serial dilutions was printed in duplicate. Prefixes t and p indicate the total and phosphorylated proteins, correspondingly. doi:10.1371/journal.pone.0013805.g002 proteins, perhaps reflecting slower dynamics of the infection, compared to the higher MOI. The third group of 7 proteins includes several protein kinases. Among them are serine/threonine kinase AKT (protein kinase B), the global regulator of survival; protein kinase R (PKR), which mediates apoptosis induced by viral double-stranded RNA (dsRNA); and the protein tyrosine kinase c-Src, which activity is regulated by tyrosine phosphorylation at two sites Y416 and Y527, but with opposite effects. Group III also includes apoptosis Figure 3. Total level of cellular proteins in RVFV-infected HSAECs. Slides stained with Sypro Ruby and normalized relative to uninfected HSAECs at 0 h.p.i. Each sample of 40 cells per dot along with seven 26 serial dilutions was printed in duplicate. Black and gray bars illustrate the MOI of 0.002 and 0.0002, correspondingly. Data represent mean values and confidence intervals of two-tail t-test (a = 0.05, n = 3). doi:10.1371/journal.pone.0013805.g003 inhibitor XIAP. In this group the phosphorylation levels showed a statistically significant, continuous decrease during the infection. The same pattern of continuous decrease was demonstrated for the total level of autophagic protein LC3B, and the pro-apoptotic proteins Bim and Puma belonging to the Bcl-2 family. Analysis of pro-apoptotic signaling and caspase activation Modulation of apoptosis is a known feature of RVFV [8]. Therefore, along with protein phosphorylation events we analyzed the levels of cleaved caspases. As indicated above, HSAECs infected with RVFV demonstrated increased levels of cleaved effector caspase-3, -6 and -7 at both MOIs; however, the level of caspase-9 cleaved at the activation sites D315 and D330 did not change significantly at both tested MOIs (Table 1, Fig. 6). The absence of significant changes in the level of caspase-9 involved in the intrinsic apoptotic pathway agrees with a large increase in the level of HSP27 protein, which is known to inhibit the apoptosome assembly and caspase-9 activation [17]. The levels of HSP70 and HSP 90, also capable of regulating the apoptosome, did not change in HSAECs infected with RVFV. Further evidence in favor of the intrinsic apoptotic pathway inhibition by the infection was revealed by the behavior of the proapoptotic mitochondrial proteins of Bcl-2 family, namely: the Bcl-2-associated protein X (Bax); the p53-upregulated modulator of apoptosis (Puma); the pro-apoptotic (Bim); the Bcl-2-interacting domain (Bid); and Bad. The latter inhibits anti-apoptotic protein Bcl-XL and induces apoptosis when it is phosphorylated on S136 by AKT. In RVFV-infected cells, Bad (S136) phosphorylation demonstrated no substantial change until 72 h.p.i. The levels of Bid and Bax did not change, however a decrease was observed for Puma and Bim (Table 1). Collectively, these results are consistent with upregulation of extrinsic apoptotic signaling in infected cells. PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e13805