Abstract
The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV)-infected swine testicular (ST) cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1), caspase-8, and heat shock protein 90 alpha (HSP90α) were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.
Highlights
Porcine transmissible gastroenteritis coronavirus (TGEV) is an animal coronavirus that causes severe gastroenteritis in young TGEV-seronegative pigs
Comparative threshold (Ct) cycle values in three independent experiments were calculated and the results indicated that the average Ct value for the TGEV N gene ranged from 25.2 to 27.5
In order to better understand the interactions between the host proteome and TGEV, we adopted an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative proteomic approach to investigate the altered cellular proteins of the swine testicular (ST) cells during TGEV infection in vitro
Summary
Porcine transmissible gastroenteritis coronavirus (TGEV) is an animal coronavirus that causes severe gastroenteritis in young TGEV-seronegative pigs. Diseased pigs often present with vomiting, dehydration, and severe diarrhea. The envelop, core, and nucleocapsid of the TGEV virion contain four major structural proteins: the nucleocapsid (N) protein, the membrane (M) glycoprotein, the small envelope (E) protein, and the spike (S) protein [5]. The M protein, which plays a central role in virus assembly by interacting with viral ribonucleoprotein (RNP) and S glycoproteins [6], is embedded within the virus membrane and interacts with the nucleocapsid, forming the core of TGEV virion. The E protein, a transmembrane protein that acts as a minor structural component in TGEV and affects virus morphogenesis, is essential for virion assembly and release [8]
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