Tandem Mass Tag-Based Quantitative Proteome Analysis of Porcine Deltacoronavirus (PDCoV)-Infected LLC Porcine Kidney Cells

Abstract

Porcine deltacoronavirus (PDCoV) is a newly emerging porcine pathogenic enteric coronavirus that can cause diarrhea, vomiting, dehydration, and a high mortality rate in piglets. At present, the understanding of PDCoV pathogenesis is very limited, which seriously hinders effective prevention and control. In this study, liquid chromatography tandem-mass spectrometry (LC–MS/MS) combined with tandem mass tag (TMT) labeling was performed to compare the differential expression of proteins in PDCoV-infected and mock-infected LLC-PK cells at 18 h post-infection (hpi). In addition, the parallel reaction monitoring (PRM) technique was used to verify the quantitative proteome data. A total of 4624 differentially expressed proteins (DEPs) were quantitated, of which 128 were significantly upregulated, and 147 were significantly downregulated. Bioinformatics analysis revealed that these DEPs were involved mainly in the defense response, apoptosis, and the immune system, and several DEPs may be related to interferon-stimulated genes and the immune system. Based on DEP bioinformatics analysis, we propose that PDCoV infection may utilize the apoptosis pathway of host cells to achieve maximum viral replication. Meanwhile, the host may be able to stimulate the transcription of interferon-stimulated genes (ISGs) through the JAK/STAT signaling pathway to resist the virus. Overall, in this study, we presented the first application of proteomics analysis to determine the protein profile of PDCoV-infected cells, which provides valuable information with respect to better understanding the host response to PDCoV infection and the specific pathogenesis of PDCoV infection.

Animal Coronaviruses Induced Apoptosis

“…Therefore, it was speculated that after PDCoV infection, host cells upregulate the expression of caspase-3, caspase-7, and caspase-8 to promote apoptosis. On the other hand, cleavage of PARP1 by caspase-3 may be used to downregulate the expression of PARP1 to accelerate the occurrence of apoptosis (Table 1) [113].…”

Tandem Mass Tag-Based Quantitative Proteomic Analysis of ISG15 Knockout PK15 Cells in Pseudorabies Virus Infection

“…The protein concentration in the extracts was determined with a BCA protein quantitation kit (Beyotime). Protein digestion was performed according to a previously described procedure [16]. Proteins in solution were reduced with 5 mM DTT at 56 • C for 30 min and alkylated with 11 mM IAA for 15 min at RT in the darkness.…”

“…The expression of PRV proteins was further monitored by IFA, and the results showed that almost all cells became infected at 24 hpi (Figure 1D). The time point when viral replication remains high is often regarded as the optimal time for proteomic analysis [16]. Therefore, in consideration of cell integrity and the high infection rate of the cells, 24 hpi was selected as the optimal time point for further proteomic analysis.…”

“…Despite years of intensive research, the mechanisms of PRV pathogenesis and immunomodulation remain largely unknown [11][12][13][14]. Accumulating evidence emphasizes that proteomic techniques have been increasingly widely used to screen the differentially expressed proteins associated with viral replication, an approach helpful for further revealing the molecular mechanism of virus-host cell interactions involved in viral pathogenesis [14][15][16].…”

Tandem mass tag-based proteomic analysis reveals cathepsin-mediated anti-autophagic and pro-apoptotic effects under proliferative diabetic retinopathy

“…Proteomics utilizes high-throughput technology to study structure and function of proteins, which facilitates understanding of pathogenesis and progression of disease at protein level [ 15 ]. Tandem mass tag (TMT) is one of the common quantitative methods of proteomics [ 16 , 17 ]. Compared with the traditional gel-based proteomic quantification approach, TMT has better reliability, accuracy, and coverage.…”