Quantitative Proteomic Analysis of BHK-21 Cells Infected with Foot-and-Mouth Disease Virus Serotype Asia 1

Abstract

Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantitatively study the host cell gene expression profile, in order to achieve an unbiased overview of the protein expression changes in BHK-21 cells infected with FMDV serotype Asia 1. The SILAC-based approach identified overall 2,141 proteins, 153 of which showed significant alteration in the expression level 6 h post FMDV infection (57 up-regulated and 96 down-regulated). Among these proteins, six cellular proteins, including three down-regulated (VPS28, PKR, EVI5) and three up-regulated (LYPLA1, SEC62 and DARs), were selected according to the significance of the changes and/or the relationship with PKR. The expression level and pattern of the selected proteins were validated by immunoblotting and confocal microscopy. Furthermore, the functions of these cellular proteins were assessed by small interfering RNA-mediated depletion, and their functional importance in the replication of FMDV was demonstrated by western blot, reverse transcript PCR (RT-PCR) and 50% Tissue Culture Infective Dose (TCID50). The results suggest that FMDV infection may have effects on the expression of specific cellular proteins to create more favorable conditions for FMDV infection. This study provides novel data that can be utilized to understand the interactions between FMDV and the host cell.

Fig 1. FMDV infection in BHK-21 cells.

(A) Photomicrographs of BHK-21 cells infected with FMDV serotype Asia1 at MOI = 1 PFU/cell or mock-infected for the indicated hours post infection (h.p.i.). Images were taken at an original magnification of 100×. (B) Confirmation of FMDV capsid proteins in the cell lysate at the indicated h.p.i. by western blot using the pig anti-FMDV Asia 1 serum as primary antibody. Actin was used as loading control.

Fig 2. Distributions of proteins identified in BHK-21 cells infected with FMDV and proteome-wide accurate quantitation and significance.

(A) The left (black red color) represents up-regulated host proteins in virus-infected cells. The right (green color) represents down-regulated host proteins based on significance B ≤ 0.05. The other parts represent unchanged proteins in FMDV-infected cells. (B) Signal intensities (log10) of all quantified proteins in the FMDV-infected cells are shown as functions of their fold change (log2). The spread of the cloud is lower at high abundance, indicating that quantification is more precise. The criterion of being identified as a significantly regulated protein can be evaluated by the significance B level indicated in blue, red, purple, and green.

Fig 3. Classification of proteins in BHK-21 cells infected with FMDV according to their assigned fraction and biological function.

(A) Biological process; (B) Cellular component; (C) Molecular functions. More information is available in Supplemental material (S1 Table and S3 Fig).

Fig 4. Ingenuity pathway analysis of proteins that were significantly altered in BHK-21 cells infected with FMDV Asia 1.

The proteins indentified were involved in Protein Synthesis, Gene Expression, Nucleic Acid Metabolism (A); Cell Death, Inflammatory Response, Infectious Disease (B); Hematological System Development and Function, Hematopoiesis, Cellular Development (C); Gene Expression, Cell Death, Cell Morphology (D); DNA Replication, Recombination and Repair, Cancer, Skeletal and Muscular Disorders (E). Proteins shaded in green indicate decrease in abundance and proteins shaded in red indicate increase in abundance in the FMDV-infected cells compared with mock-infected cells. The color intensity corresponds to the degree of abundance. Proteins in white are those known to be in the network but were not identified in our study through the Ingenuity Pathways Knowledge Base. The shapes denote the molecular class of the protein. The solid lines indicate the promotional interactions while the dashed lines indicate the suppressive interaction.

Fig 5. Confirmation of six up-regulated or down-regulated proteins by immunoblot compared to the SILAC analysis.

Relative expression of six representative proteins (normalized to actin band) was determined using image densitometry by software Image J. The ratio of I/M is reflected by the band densitometry of infection compared to that of mock infection. M, Mock; I, infected.

Fig 6. Subcellular localization of target proteins in mock- and FMDV-infected BHK-21 cells by confocal microscopy analysis.

Target proteins are stained green, FMDV proteins are shown in red, and the nuclei are stained blue with DAPI.

Fig 7. Effects of representative protein on FMDV replication in BHK-21 cells were confirmed by western blot analysis after RNA interference.

FMDV capsid protein was detected by the pig anti-FMDV Asia 1 serum. Relative expression of FMDV proteins was determined using image densitometry by software Image J. The relative quantity of FMDV protein was showed by normalization of ratio of FMDV capsid protein to actin with that ratio of normal cells. Bars represented the means±standard deviations (SD) of three independent experiments.

Fig 8. Quantification of FMDV gene in BHK-21 cells after knockdown genes of representative proteins.

The cells transfected with untargeting siRNA was set as negative control, the cells untransfected was blank control. FMDV RNA was indicated by the FMDV 3D gene which is conservative and stable in the FMDV genome. Relative quantity of gene was determined using image densitometry by software Image J. The relative quantity of FMDV gene was showed by normalization of ratio of FMDV 3D gene to actin gene with that ratio of normal cells. Bars represented the means±SD of three independent experiments.

Fig 9. FMDV titer was detected by the TCID50 after transfection with siRNA or untargeting siRNA.

The cells transfected with untargeting siRNA was set as negative control, the cells untransfected was blank control. Bars represent the means±SD of three independent experiments.