Quantitative proteomics analysis reveals BAG3 as a potential target to suppress severe acute respiratory syndrome coronavirus replication

Abstract

The discovery of a novel coronavirus (CoV) as the causative agent of severe acute respiratory syndrome (SARS) has highlighted the need for a better understanding of CoV replication. The replication of SARS-CoV is highly dependent on host cell factors. However, relatively little is known about the cellular proteome changes that occur during SARS-CoV replication. Recently, we developed a cell line expressing a SARS-CoV subgenomic replicon and used it to screen inhibitors of SARS-CoV replication. To identify host proteins important for SARS-CoV RNA replication, the protein profiles of the SARS-CoV replicon cells and parental BHK21 cells were compared using a quantitative proteomic strategy termed "stable-isotope labeling by amino acids in cell culture-mass spectrometry" (SILAC-MS). Our results revealed that, among the 1,081 host proteins quantified in both forward and reverse SILAC measurements, 74 had significantly altered levels of expression. Of these, significantly upregulated BCL2-associated athanogene 3 (BAG3) was selected for further functional studies. BAG3 is involved in a wide variety of cellular processes, including cell survival, cellular stress response, proliferation, migration, and apoptosis. Our results show that inhibition of BAG3 expression by RNA interference led to significant suppression of SARS-CoV replication, suggesting the possibility that upregulation of BAG3 may be part of the machinery that SARS-CoV relies on for replication. By correlating the proteomic data with these functional studies, the findings of this study provide important information for understanding SARS-CoV replication.

FIG. 1.

Schematic showing application of forward and reverse SILAC combined with LC-MS/MS for the comparative analysis of protein expression in SARS-CoV replicon cells (SCR-1) and their parental BHK21 cells.

FIG. 2.

Pie chart representations of the distribution of differentially expressed proteins according to their molecular functions (A) and biological processes (B). Categorizations were based on information provided by the online resource the PANTHER classification system.

FIG. 3.

Western blot and densitometric analysis of nine DEPs and internal control protein tubulin. (A) Western blot images for nine DEPs and internal control protein tubulin. (B) Relative expression of nine DEPs (normalized to tubulin band) was determined using image densitometry and expressed in a bar chart format. The error bars represent the standard deviation of the mean for three representative analyses.

FIG. 4.

Effects of BAG3 knockdown on SARS-CoV replicon cells. (A) The knockdown of BAG3 in SCR-1 cells was confirmed by Western blotting analysis. Western blotting results indicated that BAG3 is upregulated in SCR-1 cells, and knockdown of BAG3 led to reduction of SARS-CoV proteins in SCR-1 cells. (B) Inhibition of BAG3 expression led to suppressed GFP fluorescence in SCR-1 cells compared to untreated SCR-1 cells or cells transfected with control siRNA. (C) Quantification of SARS-CoV mRNA revealed markedly reduced mRNA in BAG3 knockdown SCR-1 cells compared to untreated cells or cells transfected with control siRNA.

FIG. 5.

BAG3 is essential for efficient SARS-CoV replication. Columns are the means of three independent experiments; error bars represent ± standard deviation. The asterisks indicate significant difference (P < 0.05). (A) Confirmation of BAG3 knockdown in Vero-KD cells by Western blotting analysis. (B) Quantification of SARS-CoV RNA revealed markedly reduced SARS-CoV RNA in Vero-KD cells compared to parental Vero E6 or Vero-NC cells. Quantitative PCR was performed as described in Materials and Methods. (C) The reduction in SARS-CoV or IBV titers was determined by plaque assays. Vero E6, Vero-NC, and Vero-KD cells were infected with SARS-CoV or IBV, and the plaque numbers in the different cell lines were then normalized to the number of plaques that formed in the Vero E6 cells.