Reverse-phase phosphoproteome analysis of signaling pathways induced by Rift valley fever virus in human small airway epithelial cells - PubMed (original) (raw)

Reverse-phase phosphoproteome analysis of signaling pathways induced by Rift valley fever virus in human small airway epithelial cells

Taissia G Popova et al. PLoS One. 2010.

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-κB 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

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 1.4×104 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.

Figure 2

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 2× serial dilutions was printed in duplicate. Prefixes t and p indicate the total and phosphorylated proteins, correspondingly.

Figure 3

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 2× 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 (α = 0.05, n = 3).

Figure 4

Figure 4. Confirmation of RPMA data by Western blot using antibodies against phosphorylated and total forms of several proteins.

Western blots were performed on two independent experiments. The RVFV-infected HSAECs with MOI of 0.002 and 0.0002 at corresponding time points are indicated with a + sign. Samples 1, 2, 4, 6, 8 and 11 are from untreated cells at 0, 5, 24, 30, 48 and 72 h.p.i. and samples 3, 5, 7, 10 and 13 are RVFV-infected HSAECs with MOI of 0.002 at 5, 24, 30, 48 and 72 h.p.i. Samples 9 and 12 correspond to the cells infected with MOI of 0.0002 at 48 and 72 h.p.i.

Figure 5

Figure 5. Comparison of RPMA and Western blot data.

Left panels (A) show the RPMA data. Right panels (B) show Western blot confirmatory data. X axis labels, time post infection (h.p.i.); Y axis labels, protein level relative to untreated cells (%) at 0 h.p.i. Open triangles, closed triangles and open squares correspond to the low MOI, high MOI and uninfected cells, respectively. RPMA data represent mean values and confidence intervals of two-tail t-test (α = 0.05, n = 3). Western blot data represent average of two independent experiments.

Figure 6

Figure 6. RPMA data for caspase-3, -6, -7, and -9 and actin.

X axis labels, time post infection (h.p.i); Y axis labels, protein level relative to untreated cells (%) at 0 h.p.i. Open triangles, closed triangles and open squares correspond to the low MOI, high MOI and uninfected cells, respectively. Data represent mean values and confidence intervals of two-tail t-test (α = 0.05, n = 3).

Figure 7

Figure 7. Effect of inhibitors on RVFV replication.

HSAECS were plated at 5×104 cells per well in a 96-well plate and pretreated with either DMSO or indicated inhibitors for 5 h prior to RVFV infection at MOI of 0.002 as described in Materials and Methods. Following infection, fresh medium containing inhibitors was added. Supernatants were collected at 48 h.p.i., viral RNA extracted and analyzed by q-RT-PCR. * p = 0.0002, ** p = 0.031 (t-test) relative to mock control.

Figure 8

Figure 8. RPMA data presented as a diagram of signaling events in HSAECs infected with RVFV.

Among the most prominent responses was activation of MAP kinases (p38, JNK and ERK) and the downstream transcriptional factors [STAT1 (Y701), ATF2 (T69–71), MSK1 (S360), and CREB (S133)]. STAT3 and NF-κB phosphorylation were also increased. Activation of p53(S15, S46), which typically accompanies transcriptional upregulation of the apoptotic genes correlated with the increased levels of cleaved effector caspase-3, -6 and -7 indicating activation of the extrinsic apoptotic pathway. RVFV infection also downregulated phosphorylation of a major anti-apoptotic regulator of survival pathways, AKT (S473). Consistent with this, the level of apoptosis inhibitor XIAP100 was decreased. The intrinsic apoptotic pathway marker, caspase-9, demonstrated only a marginal activation accompanied by increased level of the inhibitor of apoptosome formation, HSP27.

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