Analysis of Interaction Network Between Host Protein and M Protein of Swine Acute Diarrhea Syndrome Coronavirus

Abstract

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enterovirus that can cause acute diarrhea and death in piglets and cause serious economic losses to the pig industry. SADS-CoV membrane (M) protein mainly plays a key role in biological processes, such as virus assembly, budding, and host innate immune regulation. Understanding the interaction between M protein and host proteins is very important to define the molecular mechanism of cells at the protein level and to understand specific cellular physiological pathways. In this study, 289 host proteins interacting with M protein were identified by glutathione-S-transferase (GST) pull-down combined with liquid chromatography-mass spectrometry (LC-MS/MS), and the protein-protein interaction (PPI) network was established by Gene Ontology (GO) terms and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways analysis. Results showed that SADS-CoV M protein was mainly associated with the host metabolism, signal transduction, and innate immunity. The Co-Immunoprecipitation (CO-IP) validation results of six randomly selected proteins, namely, Rab11b, voltage-dependent anion-selective channel 1 (VDAC1), Ribosomal Protein L18 (RPL18), RALY, Ras Homolog Family Member A (RHOA), and Annexin A2 (ANXA2), were consistent with LC-MS results. In addition, overexpression of RPL18 and PHOA significantly promoted SADS-CoV replication, while overexpression of RALY antagonized viral replication. This work will help to clarify the function of SADS-CoV M protein in the life cycle of SADS-CoV.

Keywords: RALY; RHOA; RPL18; membrane (M) protein; protein interaction network; swine acute diarrhea syndrome coronavirus (SADS-CoV).

Figure 1

Identification of interaction factors between Swine acute diarrhea syndrome coronavirus (SADS-CoV) M protein and host. A large number of glutathione-S-transferase (GST)-M recombinant proteins were expressed and purified by magnetic beads with GST fusion protein for purification and GST pull-down experiments. Then sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for isolation and identification. M: page ruler protein marker; Lane 1: purified GST-M fusion protein elution solution; 2: GST protein pull-down result; 3: GST-M protein pull-down result.

Figure 2

The PPI network generated in this study. Construction and analysis of the protein-protein interaction (PPI) network using the STRING database. The network of swine acute diarrhea syndrome coronavirus (SADS-COV) M-interacting proteins interacting was constructed and plotted using the network analyzer tool, Cytoscape v.3.8.2. The nodes are identified host proteins, expressed as their respective NCBI gene names. Edges represent the interaction between nodes. The higher the degree, the larger the node and the darker the color.

Figure 3

Gene ontology (GO) analysis based on the interaction between cell protein and M. Using Cytoscape v.3.8.2 and ClueGO software plug-in, the GO distributions of all proteins were divided into three types. The y was shown for significantly enriched terms based on biological process, molecular function (MF), and cellular component at p < 0.05.

Figure 4

KEGG pathway enrichment analysis. The enriched pathways targeted by Swine acute diarrhea syndrome coronavirus (SADS-COV)-M-interacting proteins were analyzed using the Kyoto Encyclopedia of Gene and Genomes (KEGG) functional annotation pathway database. The terms that were significantly enriched (p < 0.05) were shown.

Figure 5

Validation of M-host protein interactions. IPI-2I (A,B) and Vero (C,D) cells were co-transfected with plasmids expressing Flag-Rab11b, Flag-VDAC1, Flag-RPL18, Flag-RALY, Flag-RHOA, or Flag-ANXA2 and plasmids expressing HA-M, respectively. Among them, HA-M or Flag-Rab11b co-transfected with empty vector were served as negative controls, respectively. The cell lysates were immunoprecipitated with FLAG (A,C) or HA (B,D) beads, anti-Flag, or anti-HA mAbs, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western-blotted and detected with corresponding antibodies, respectively. GAPDH was served as an internal loading control.

Figure 6

Overexpression of host proteins RPL18, RALY, and RHOA affects swine acute diarrhea syndrome coronavirus (SADS-COV) replication. (A) When the Vero cells were laid on 12-well plates, and the confluence degree reached 80%, empty vector (4 μg/well), Flag-RPL18 (4 μg/well), Flag-RALY (4 μg/well), and Flag-RHOA (4 μg/well) were transfected into the cells. At 24 h after transfection, 1 multiplicity of infection (MOI) dose of SADS-COV was added to the cell. Viral titers were detected by TCID50 at 36 hpi. (B) Sample treatment as described in (A). After 36 hpi, the samples were collected by RNAiso Plus, and the total RNA was extracted. The mRNA expression level of SADS-CoV N gene was evaluated by real-time RT-qPCR. Flag-RHOA (C), Flag-RALY (D), and Flag-RPL18 (E) were transfected into Vero cells at different doses (1, 2l, and 4 μg/well) with empty vector, and the rest were treated as described in (A). N protein expression was detected by Western blotting at 36 hpi. GAPDH was used as an internal loading control. The band densitometry was analyzed by Image J software. All data are presented as means ± SD and were analyzed by GraphPad Prism software (GraphPad Software, San Diego, CA, USA) using a t-test (*0.05 < p < 0.01, **p < 0.01).