FIG 1.

Quantitative proteomics analysis of SFTSV-infected HEK 293 cells. (A) Kinetics of SFTSV replication in HEK 293 cells. HEK 293 cells were infected with SFTSV at an MOI of 5, the supernatants were harvested at the indicated time points, and the virus titers were measured by determining the TCID₅₀. All experiments were performed at least three times, and values represent means ± the SDs from three replicates. (B) HEK 293 cells were infected with SFTSV at an MOI of 5, and at the indicated time points HEK 293 cells were harvested and subjected to MTT assay to measure cell viability. (C) Workflow for iTRAQ-based quantitative proteomic analysis of SFTSV-infected HEK 293 cells. (D) Volcano plot showing log₂-fold change plotted against the –log₂-adjusted P value for SFTSV-infected cells versus mock-treated cells at different times p.i. (E) Kinetics of the viral protein ratio and infection ratio of SFTSV-infected HEK 293 cells. The viral protein ratio was measured by MS. The infection ratio was measured by detecting NP-positive cells versus all cells detected. (F) Validation of MS results using quantitative RT-PCR. HEK 293 cells were infected with SFTSV at an MOI of 5 or mock infected. At indicated time intervals, cells were harvested, and intracellular mRNAs were extracted and subjected to reverse transcription. The intracellular RNA levels of the corresponding proteins were measured with quantitative RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin, and actin were chosen as internal controls. Intracellular RNA levels at each time point of SFTSV infection were normalized to those in the mock-infected cells. The experiments were repeated twice. Bars in panels A and D represent the SD. M, mock treated; V, SFTSV infected; N, number of proteins quantified; up, upregulated protein; down, downregulated protein; FN1, fibronectin; GOLIM4, Golgi integral membrane protein 4; JUN, transcription factor AP-1; RIOK2, serine/threonine protein kinase RIO2; RNF25, E3 ubiquitin-protein ligase RNF25; TEAD1, transcriptional enhancer factor TEF-1; TSC22D4, TSC22 domain family protein 4; VAPA, vesicle-associated membrane protein-associated protein A.