MDBK cells are currently widely used in the study of BVDV, and few researchers have studied the proteomics of BVDV infected MDBK cells. The research and development of BVDV is in a relatively lagging state. By studying the differentially expression proteins of BVDV-infected MDBK cells in the early, middle and late stages, we can systematically understand the interaction between the virus and host cells, looking for host cell immune regulation related proteins to BVDV infection. Furthermore, this study can also provide new ideas for the prevention and control of BVDV and a reference for the research of other viruses of the genus Pestivirus. So far, BVDV has lots of various subtypes through research globally [13]. Because of the difficulty of studying all these types, we choose BVDV-1 NADL, commonly used in the Chinese stock raising industry [14].
Currently, various proteomic methods have been widely used to study the interaction between virus and host cell [8, 15]. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) is a peptide in vitro labeling technology developed by American Applied Biosystems ABI. As a new proteome quantification technology, iTRAQ can perform more accurate and effective high-throughput proteome analysis than traditional protein qualitative technology. In recent years, proteomics technology based on iTRAQ has been applied to virology research many times.
The interaction existing in virus and host cell is a very complex course, involving a large number of protein expression changes and signal transduction [16]. Here, iTRAQ coupled with tandem mass spectrometry (LC-MS/MS) was used to get an analysis of the protein expression profiles of MDBK cells which were infected with BVDV NADL at 3 time points. We found that 357, 101, and 66 DEPs were seen at 12, 24, and 48 hpi based on a fold change > 1.5 or < 0.67 and p value < 0.05. From our results, a significant difference was found in proteins in host cells. It means that the DEPs play a significant guiding part in BVDV NADL-infected MDBK cells. Massive proteins were up-regulated in each period. Simultaneously, the up-regulated proteins were displayed much more than the down-regulated proteins, and this appears clearly at 48 hpi. This suggests the possibility for the host at different time points to be against BVDV NADL infection. Further knowledge of the function of these proteins can help to understand the relationship between MDBK cells and BVDV NADL. It provides a theoretical basis for the response of host cells to BVDV NADL infection and the development of vaccine.
According to the analysis of these dysregulated proteins with the GO annotation, we found that GO terms were completely different at 12 hpi, 24 hpi and 48hpi in order. It also showed that infected cells respond differently at various time points of BVDV NADL infection. Actually, a large number of the differentially expressed proteins are concentrated in the regulation of biological process (BP) and cellular process and signal transduction at 12 hpi. While at 24 hpi, the most enriched GO terms were cell surface receptor signaling pathway, intermediate filament and extracellular space. However, in the later stage of infection, there was no significant enrichment of differentially expressed proteins in the biological process. GO annotation and pathway analysis revealed that these proteins are being in a variety of cellular biological processes, containing metabolic pathways, protein processing in the endoplasmic reticulum, ECM receptor interactions, and actin cytoskeleton rules. By protein interaction network analysis, we speculate that the MAPK signaling pathway may play a role in BVDV NADL-infected cells. These results of this study lay a foundation for further research on the pathogenesis of BVDV and the selection of antiviral targets.
1. Differentially expressed proteins involved in immunity system
Non-specific immunity system refers to the body's innate normal physiological defense function, which can make corresponding immune responses to the infection of various viruses. Pattern recognition receptors (PRRs) are the representative of immune receptors in innate immunity and can recognize viral RNA in the cytoplasm [17]. It actually plays a role in anti-viral natural immunity [18]. In this study, lots of the DEPs were being in the Toll-like receptor(TLR), nucleotide binding oligomerization domain (NOD)-like receptor (NLR) and MAPK signaling pathways, including ring finger protein 31 (RNF31), ANTXR cell adhesion molecule 2 (ANTXR2), tumor protein p53 binding protein 1 (TP53BP1), caspase 8 (CASP8), and insulin receptor (INSR). The MAPK family plays a regulatory role in physiological and pathological processes such as cell growth, differentiation and inflammation. And many studies have shown that the RLR (retinoid acid-inducible gene-Ι(RIG-I)-like receptor), TLR and NLR signaling pathways play significant regulatory roles in Flavivirus-infected cells [19, 20].
We found that the expression of some proteins was changed after BVDV NADL infection such as interferon regulatory factors (IRFs). IRFs are a kind of multifunctional transcription factors, which can specifically bind to interferon (IFN) gene promoter and interferon stimulatory response elements (ISRE) in interferon stimulation response gene (ISG). Therefore, IRFs have important significance in the body's anti-viral infection and regulating cell growth. From our research, the data suggested that the expression of interferon regulatory factor 2 (IRF2) were meaningfully upregulated. IRF2 is reported to be an important regulator of breast cancer invasion and metastasis; however, it is needed to explore its function in the antiviral process and immune regulation in further studies.
2. Differentially expressed proteins involved in cytoskeleton and ECM-receptor interactions
Cell morphology, cell movement and intercellular adhesion are maintained by cytoskeletal proteins. In addition, the cytoskeleton also plays a key role in organelle location and vesicle transport. It is worth mentioning that the cytoskeletal network also plays the role of promotion in the process of virus infection. After the host cell is infected by the virus, its cytoskeleton will break or even disintegrate [21]. Of the 8 DEPs in the regulation of actin cytoskeleton, integrin subunit beta 5 (ITGB5), ENAH actin regulator (ENAH) and Rho guanine nucleotide exchange factor 7 (ARHGEF7) are significantly up-regulated.
In the present study, integrin subunit beta 5 (ITGβ5), collagen type IV alpha 1 chain (COL4A1) and syndecan 1 (SDC1) are significantly up-regulated. The integrin acts as a transmembrane joint between the extracellular matrix cell and the intracellular actin skeleton, which make the extracellular matrix cell connected with the intracellular skeleton network into a whole. A series of related studies have shown that the abnormal expression of ITGβ5 is closely related to the pathological process of various diseases. ITGβ5 can promote the formation of blood vessels in various diseases. It can also interact with endothelial growth factor receptor 2 (VEGF2) to inhibit the apoptosis of cells. Collagens are the most abundant and widely distributed functional proteins in mammals and play a significant role in cell movement, angiogenesis, and tissue formation or repair. Collagens are also main components of the ECM which were associated with many other diseases, including Ehlers-Danlos syndrome, Kniest dysplasia, Alport syndrome, and certain arterial aneurysms [22]. Even if the different shapes of collagens have many different kinds of functions in BVDV NADL infection, that is still not clear. After BVDV NADL-infection, we found a lot of significant pathological changes in the cells with the help of viral titer at 48 hpi. However, the experimental results do not indicate that the proteins related to the cytoskeleton and ECM-receptor interactions in MDBK cells could promote the proliferation and release of BVDV NADL.
3. Differentially expressed proteins involved in endoplasmic reticulum protein processing
Endoplasmic reticulum (ER) is the most important part for protein synthesis and maturation. The endoplasmic reticulum is found in many molecular chaperones and helps proteins fold and assemble [23]. Lots of studies have shown that viral infection not only alters the endoplasmic reticulum, but also activates the unfolded protein response (UPR), thereby promoting viral replication [24, 25]. In our study, 14 DEPs were in endoplasmic reticulum protein processing. Among these proteins, endoplasmic reticulum lectin 1 (ERLEC1 ) and lectin and mannose binding 1 (LMAN1 ) are both up-regulated at 48 hpi. Moreover, ubiquitin conjugating enzyme E2 J1 (UBE2J1) is up-regulated at 12 hpi. The results showed that DnaJ heat shock protein family (Hsp40) member A2 (DNAJA2), heat shock protein family A (Hsp70) member 8 (HSPA8) and heat shock protein family A (Hsp70) member 1A (HSPA1A) are down-regulated at 24 hpi, while DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) is significantly up-regulated at 12hpi. Heat shock protein (HSP) is a kind of highly conserved protein family whose expression is inhibited by the body or cells under normal conditions and stimulated by adverse environmental factors [26]. It has various biological functions including cell protein self-stabilization, anti-apoptosis, anti-oxidation, cooperative immunity, etc. For example, HSP70 is highly expressed in various DNA virus and RNA virus infections and inhibits virus replication [27, 28]. The decreased activity of heat shock proteins will lead to the development of inherited peripheral motor neuropathy, Alzheimer's disease and other diseases [29]. This study identified that HSPA8 and HSPA1A were significantly down-regulated at 24 h after BVDV NADL infection, which may cause the host to display abnormal neurological symptoms. Since heat shock proteins are involved in protein folding, the activation of UPR signals in the endoplasmic reticulum stress response may play a role in specific viral functions. Therefore, endoplasmic reticulum stress proteins may also be important for the replication of many viruses [25]. The entry of the virus into the target cell is one of the most important steps in virus reproduction, so further study is needed to verify the specific role of these differential proteins after BVDV NADL infection.
4. Differentially expressed proteins involved in translation
Most translation-related proteins are up-regulated during BVDV NADL infection. Among the ribosomal proteins, Treacle ribosome biogenesis factor 1(TCOF1), nuclear transport factor 2 like export factor 2(NXT2), ribosomal protein L37(RPL37), ribosomal protein L29 (RPL29) and mitochondrial ribosomal protein S18A(MRPS18A) are all up-regulated in the early stages of infection. As the translation initiation factor, eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), eukaryotic translation initiation factor 4E (EIF4E) and eukaryotic translation initiation factor 3 subunit F (EIF3F) are up-regulated in the course of BVDV NADL-infection. We hypothesized that BVDV NADL would use the MDBK cells' protein synthesis system to produce substantial viral proteins.
5. Differentially expressed proteins involved in metabolic processes
Studies have shown that cell metabolism in host cells can be significantly changed by viral infection [30, 31]. In our research, we found many proteins which were involved in metabolic processes expressed variously in the BVDV NADL-infected MDBK cells. And 59 differentially expressed proteins (9.14%) were sorted as metabolic pathways. These proteins are mainly involved in glycolysis, pyruvate metabolism, and citrate cycle (TCA cycle). Among these proteins in the metabolic pathways, pyruvate dehydrogenase E1 beta subunit (PDHB), aldehyde dehydrogenase 7 family member A1 (ALDH7A1), aldehyde dehydrogenase 3 family member A2 (ALDH3A2) and pyruvate kinase M1/2 (PKM) are involved in both the glycolysis and pyruvate metabolism. Aldehyde dehydrogenase 7 family member A1 (ALDH7A1) and pyruvate kinase M1/2 (PKM) are down-regulated at 24 hpi. Pyruvate dehydrogenase E1 beta subunit (PDHB), citrate synthase (CS), ATP citrate lyase (ACLY) and isocitrate dehydrogenase (NAD(+)) 3 non-catalytic subunit gamma (IDH3G) are involved in the citrate cycle (TCA cycle). At present, the interaction mechanism of virus-host metabolism needs to be further studied. In particular, how the key genes of the metabolic pathway regulate viral susceptibility is worth considering. This process provides a new direction for this study. Antiviral drugs can be studied by studying how protein expression in metabolic pathways inhibits virus generation.