Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection

Abstract

Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase protein array (RPPA) to define changes in signaling cascades after in vitro infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.

Fig 1. Viral kinetics of virulent and attenuated RVFV viruses.

A) Schematic depicting infection and harvest conditions for comparative analysis. HSAECs were infected with attenuated (MP12) or virulent (ZH548) RVFV at an MOI 5 using conditioned media. RVFV RNA replication (B), percentage of RVFV NP positivity (C), and infectious viral titers (D) were determined at the indicated time points (hours post infection (hpi)). The data are plotted as means with standard deviations: for panel (B) n = 3, (C) n = 6 and (D) n = 9. ** = P≤0.01 and **** = P≤0.0001.

Fig 2. RPPA and Western blot confirmation of Smad protein phosphorylation.

A) Phosphorylation status of nine RPPA targets after RVFV infection of HSAECs. Bar graph represents the mean fold change with standard deviation of three independent replicates. Phospho-specific Smad antibodies are noted as: pSmad1/5/9 MH2 [phosphorylated Smad1/5 (S463/465)/9 (S465/467)], pSmad1/5 MH2 [phosphorylated Smad1/5 (S463/465)], pSmad2 MH2 [phosphorylated Smad2 (S465/467)], and pSmad2 L [phosphorylated Smad2 (S245/250/255)]. Statistical significance of the phosphorylation changes are highlighted in Table 1. HSAEC (B) and Huh-7 cells (C) were infected at an MOI 5 with MP12 or ZH548, or mock infected (media alone). Protein lysates harvested at 3, 9, and 18hpi were analyzed for pSmad1/5/9 MH2, pSmad1/5 MH2, pSmad2 MH2, pSmad2 L, RVFV NP, Actin as well as total protein levels for Smad1, Smad5, and Smad2 by western blotting. Blots represent one replicate of three independent experiments. M:Mock; MP:MP12; ZH: ZH548.

Fig 3. Smad phosphorylation is dependent on RVFV replication and NSs expression.

A) Analysis of HSAECs infected with MP12, ZH548 or their UV-inactivated (UVin) controls at 9 and 18hpi. Protein lysates were analyzed by western blot for pSmad1/5/9 MH2, pSmad1/5 MH2, pSmad2 MH2, pSmad2 L, RVFV NP, actin as well as total protein levels for Smad1 and Smad2. M: Mock; MP: MP12; ZH: ZH548. B) Smad phosphorylation in HSAECs infected with increasing MOIs (0.1, 0.5, 1, and 5) of MP12 virus at 9hpi was examined as described in panel A. C) Analysis of Vero cells infected at an MOI 3 with MP12 or MP12 ΔNSs. With the exception of pSmad1/5/9 MH2, protein lysates were analyzed by western blot as described in panel A. D) Vero cells transfected with media alone, pcDNA3.1 vector (2.5 μg), or pcDNA3.1-Flag-NSs (2.5 μg) were harvested at 24 hours post transfection. Protein lysates were analyzed by western blot for pSmad1/5 MH2, pSmad2 MH2, pSmad2 L, Flag-NSs as well as total protein levels for Smad1 and Smad2.

Fig 4. siRNA knockdown of Smad1, -2 and 4 does not impact RVFV replication.

HSAECs transfected with media alone (Mock) or siRNAs (75nM) directed against a nontargeting control (NTC), Smad1, -2, or -4 were infected with MP12 virus (MOI 0.1). Both extracellular media supernatants (A) and protein lysates (B,C) were harvested at 9 and 24hpi. A) Infectious viral titers were determined by plaque assay. Data plotted as a bar graph of the mean with standard deviation of four replicates. Protein lysates from single (B) and double knockdowns (C) were analyzed by western blot for actin, total Smad1, -2, and -4 expression.

Fig 5. Nuclear localization of phosphorylated Smad proteins.

HSAEC cells either alone (Mock), treated with TGF-β3 (50ng/mL, 2hrs), BMP-4 (50ng/mL, 2hrs), or infected with MP12 (MOI 5) for 9 or 18hpi were cell fractionated. 10ug of nuclear extracts (NE) or cytoplasmic extracts (CE) were probed for pSmad1/5 MH2, pSmad 2 MH2, GAPDH and Lamin A/C levels.

Fig 6. Promoter analysis of RVFV RNASeq yields potential Smad-dependent promoters.

A) Schematic depicting data-mining of RVFV RNASeq dataset for Smad-dependent promoters. B) Top ten significantly altered canonical pathways from IPA are shown for the virulent RVFV strain at 18hpi. The top axis indicates the statistical significance calculated using the right-tailed Fisher exact test. Threshold line represents significance cut-off at p = 0.05. Bars represent the level of significance, with orange and blue bars indicating whether the pathway is predicted to be activated or inhibited, respectively (z-score). Pathways where no prediction can be made are colored gray. The ratio (bottom axis) represents the number of molecules identified in a given pathway divided by total number of molecules that constitute that pathway.

Fig 7. ChIP analysis of possible Smad-dependent transcripts.

Lysates from mock, TGF-β activated, and MP12 infected were utilized for Smad4 (A) or methylated histone H3 lysine 4 (mH3K4; B) immunoprecipitations. Relative DNA abundance from immunoprecipitations was determined by qPCR. Signal over background normalization and fold enrichment was calculated for each condition. Bars represent means and standard deviations of four replicates. C) Levels of IL1R2, IL1RL1, and VAV3 RNA are analyzed by qRT-PCR. RNA lysates from HSAECs infected with MP12 (blue) or ZH548 (red) RVFV at an MOI 5 were utilized. Bars represent means and standard deviations of four replicates.