Proteomics is a novel methodology to detect components of cellular protein interactions as well as host cellular pathophysiological processes that occur during virus infection . Until present investigation, no results have been reported for performing analysis of differential proteome of host cells infected with TGEV. In this study, 2D DIGE coupled with MALDI-TOF/TOF was used to analyze the differential proteome of ST cells infected with TGEV. The 33 differential protein spots were successfully identified as 17 proteins, of which function in diverse biological processes.
Cytoskeletal protein expression was altered in TGEV-infected ST cells. The cytoskeleton filaments are dynamic and divided into three types: microfilaments (actin filament), microtubules, and intermediate filaments . The intermediate filaments can provide mechanical stability to cells, while actin and microtubule cytoskeletons are responsible for trafficking of numerous endogenous cargos as well as intracellular microorganisms throughout the cells . Many viruses use the cytoskeleton for infection and replication, such as HIV-1 . In present study, differentially expressed microfilament-associated proteins beta actin and alpha tropomyosin, microtubule-associated alpha tubulin and beta tubulin, as well as the intermediate filament-associated vimentin and keratin 19 were identified (Table 1), indicating that TGEV infection and replication involves cellular skeleton.
The actin and microtubule cytoskeleton play important roles in the life cycle of viruses . Numerous viral proteins interact with actin-binding proteins or directly with actin . Microtubules and microtubule-associated proteins are known to play important roles in intracellular trafficking of viral components as well as virions in the infected host cell . In this study, the up-regulated microfilament-associated proteins beta actin and microtubule-associated alpha tubulin and beta tubulin were identified, which was believed to facilitate the transport of viral proteins of TGEV from rough endoplasmic reticulum (ER) and Golgi apparatus to the reservoir for viral replication.
Vimentin is a major component of type III intermediate filaments found in many cell lines . This protein serves to maintain cell shape and is involved in attachment, migration and cell signaling . Major changes in the distribution of vimentin are observed when the cell moves and divides , but this protein is also redistributed in cells expressing misfolded proteins and during virus infection . Previous studies have shown that intermediate filament protein vimentin was cleaved by human immunodeficiency virus type 1 protease (HIV-1 PR)  and that vimentin networks collapsed and was dispersed in IBDV-infected cells . In present study, 8 differential protein spots were identified as vimentin including 5 up-regulated protein spots and 3 down-regulated protein spots. Further study is required to determine whether TGEV papain-like protease 1 (PL1pro)  cleaves vimentin into different isoform or subunit, using a similar strategy as HIV.
Among those differentially expressed host proteins, some are known to participate in viral replication and translation (Table 1). Positive-strand RNA viruses must recruit normal components of host cellular RNA processing or translation machineries for viral RNA synthesis and protein synthesis . Heterogeneous nuclear ribonucleoprotein U (HNRNPU) is an abundant, strictly nuclear phosphoprotein that interacts directly with RNA through a carboxy-terminal RGG sequence . HNRNPU is known to influence pre-mRNA processing, mRNA transportation to cytoplasm, intracellular localization, translation, and turnover of mRNAs . Previous studies have shown that the levels of HIV-1 viral transcripts are dramatically down-regulated in cytoplasm of infected cells by HNRNPU . In this study, the down-regulated HHRNPU was found after TGEV infection, which may facilitate the replication of viruses. Another RNA processing protein, thiopurine S-methyltransferase (TPMT), was found to be more abundant in TGEV-infected cells. TPMT is a cytoplasmic transmethylase present in prokaryotes and eukaryotes, which has a molecular mass of 28 kDa and comprises 245 amino acids . TPMT is a drug-metabolizing enzyme widely expressed in mammalian and non-mammalian cells . Previous works have reported that TPMT plays a role in BVDV virus replication and thiopurines inhibit bovine viral diarrhea virus production in a TPMT-dependent manner . Based on these data, an up-regulation of TPMT in TGEV-infected ST cells suggests that this host protein plays an important role in TGEV biology, making it possible target for future drug development.
Viruses rely on the cellular translation machinery to translate their own proteins, which facilitates the rapid production of viral proteins and renders an inhibitory effect on the production of host proteins, including host anti-viral proteins . Translation factors have been well documented as playing crucial roles in viral RNA and protein synthesis . In herpes simplex virus type 1 (HSV-1) infected HeLa cells, the synthesis of several ribosomal proteins and their assembly into ribosomes continue in spite of a general inhibition of cellular protein synthesis . Acidic ribosomal protein P0 (RPLP0) is located in the active part of the ribosome particle, at which mRNAs, tRNAs and translation factors interact during protein synthesis . The cellular RPLP0 was observed as up-regulated after virus infection, such as pseudorabies virus (PrV) infected bovine kidney cells , and Epstein-Barr virus (EBV) infected primary B cells . In this study, the up-regulated 60S RPLP0 was also found after TGEV infection, suggesting the ribosomal protein plays an important role in the translation of TGEV viral proteins.
Viruses may inhibit host protein synthesis by targeting multiple steps in the gene expression process via various pathways, for instance, the vesicular stomatitis virus (VSV) M protein inhibits the initiation of the transcription of host genes  and the SARS-CoV spike protein inhibits host cell translation by interaction with eIF3f . In this study, the up-regulated eukaryotic initiation factor 4A-I (EIF4A1) was identified after TGEV infection. The translation initiation proteins observed in TGEV infected ST cells may be a reflection of translation regulation mechanisms exploited by TGEV virus, interfering with cellular protein synthesis and translation initiation of the host cell for beneficial reasons, which need to be further studied.
Protein phosphatase 2A (PP2A) is an evolutionarily conserved enzyme that represents a major portion of serine/threonine phosphatase activity in cell extracts . PP2A enzymes have been clearly involved in regulation of cell transcription, cell cycle and viral transformation . Up-regulation of PP2A scaffold subunit A and subsequent dephosphorylation of Tyr-307 in the catalytic subunit was found, suggesting PP2A activation in Huh7 infected cells [51, 52]. Activation of serine-threonine PP2A was found in Huh7 cells upon HSV-1 infection, and PP2A activation paralleled dephosphorylation and inactivation of the downstream mitogen-activated protein (MAP) kinase pathway . In this study, the up-regulation of PP2A was found in TGEV infected ST cells, suggesting PP2A plays an important role in the dephosphorylation of cellular and viral protein during TGEV infection.
Several proteomics analysis about coronavirus had been done including SARS-CoV , IBV [55, 56], and MHV . By comparing the finding proteins in this study to previous findings, there is no common gene of target related to coronavirus. The findings in TGEV infected ST cell might not reflect the interaction between the virus and pig intestine epithelial cell. It is surprising that the proteome responses observed did not reveal any immune responses related proteins in TGEV-infected ST cells. It may be related to the host cells chosen or the fact that attenuated TGEV strain was used. Identification of cellular proteome in pig intestine epithelial cell or immune cells infected with TGEV need to be further studied.