Network of Interactions between the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus and Host Proteins

Abstract

Atypical porcine pestivirus (APPV) can cause congenital tremor type A-II in neonatal piglets, posing a significant threat to swine herd health globally. Our previous study demonstrated that the Mut domains, comprising 112 amino acids at the N-terminus, are the primary functional regions of the E2 protein of APPV. This study identified 14 host cellular proteins that exhibit potential interactions with the Mut domains of the E2 protein using yeast two-hybrid screening. Using bioinformatics analysis, we discovered that the Mut domains of the E2 protein might exert regulatory effects on apoptosis by modulating energy metabolism within the mitochondria. We also conducted co-immunoprecipitation, glutathione S-transferase pull-down, and immunofluorescence assays to confirm the interaction between the Mut domains of the E2 protein and cathepsin H and signal sequence receptor subunit 4 (SSR4). Ultimately, SSR4 enhanced APPV replication in vitro. In summary, our study successfully elucidated the interactions between the Mut domains of the E2 protein and host cell protein, predicted the potential pathways implicated in these interactions, and demonstrated SSR4 involvement in APPV infection. These significant findings contribute valuable knowledge toward a deeper understanding of APPV pathogenesis and the role of the Mut domains of the E2 protein in this intricate process.

Keywords: APPV; E2 protein; Mut domains; SSR4; protein interaction.

Figure 1

Network of cellular proteins. The network of host proteins interacting with the Mut domains of the E2 protein of APPV was constructed based on the STRING 9.0 database. APPV, atypical porcine pestivirus.

Figure 2

Enrichment analysis of the host proteins interacting with the Mut domains of the E2 protein of APPV using GO (A) and KEGG (B) databases. p < 0.05 was considered significant enrichment. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 3

The Mut domains of the E2 protein co-localize with CTSH (A) and SSR4 (B) proteins in PK15 cells. To achieve this, plasmids expressing Flag-SSR4 or Flag-CTSH, along with plasmids expressing EGFP-Mut or EGFP, were co-transfected into the cells. After 48 h, the cells were fixed with 4% paraformaldehyde, stained with mouse anti-Flag (Red), and DAPI (Blue), and subsequently analyzed using a fluorescence microscope. Green indicates the EGFP-Mut or EGFP proteins. Yellow indicates colocalization of the Mut domains of the E2 protein co-localize with CTSH or SSR4 proteins in the overlay image.

Figure 4

Interaction between the Mut domains of the E2 protein of APPV and CTSH (A) and SSR4 (B) proteins identified using a co-immunoprecipitation assay. HEK-293T cells were co-transfected with pEGFP-Mut and plasmids expressing Flag-SSR4 or Flag-CTSH. The cells were harvested after 24 h of transfection, and their lysates were subjected to immunoprecipitation using GFP antibodies, followed by immunoblotting with anti-Flag antibodies. HEK-293T, human embryonic kidney 293T; CTSH, cathepsin H; SSR4, signal sequence receptor subunit 4; APPV, atypical porcine pestivirus; GFP; green fluorescent protein.

Figure 5

Interaction between the Mut domains of the E2 protein of APPV and CTSH (A) and SSR4 (B) proteins was identified using a GST pull-down assay. The GST-Mut recombinant proteins were obtained from prokaryotic cells, purified using GST beads, and subsequently incubated with Flag-SSR4 or Flag-CTSH expressed in HEK-293 T cells. After washing, the eluted complexes were analyzed using WB assay with anti-Flag antibodies. CTSH, cathepsin H; SSR4, signal sequence receptor subunit 4; APPV, atypical porcine pestivirus; WB, Western blotting; HEK-294T, human embryonic kidney 293T; GST, glutathione S-transferase.

Figure 6

Effects of SSR4 on APPV replication. PK15 cells were transfected with pCMV-tag4A-SSR4 and pCMV-tag4A. After 24 h of transfection, the cells were infected with APPV at an MOI of 0.1. Subsequently, the cells were harvested at 24 and 48 hpi. The expression of exogenous SSR4 (A) and NS5 (B) of APPV was assessed using WB assay. The chart (C) shows the quantification of NS3/GAPDH in (B). Additionally, PK15 cells were co-transfected with non-targeting control siRNA (NC) or SSR4-targeting siRNAs. The inhibition efficiency of these three siRNAs was determined using quantitative real-time polymerase chain reaction (qRT-PCR) (D) and WB assay (E). The chart (F) shows the quantification of SSP4/α-Tubulin in (E). Furthermore, PK15 cells were transfected with NC or #1, followed by infection with APPV at an MOI of 0.1 after 24 h of transfection. Next, the cells were harvested at 24 and 48 hpi. WB analysis was conducted to determine the expression level of NS5 protein of APPV (G). The analysis of SSR4 silencing on the expression level of NS5 protein of APPV (H). Data presented are from three independent experiments and presented as mean ± SD. * p < 0.05, ** p < 0.01 and **** p < 0.0001. SSR4, signal sequence receptor subunit 4; Western blotting, WB; APPV, atypical porcine pestivirus; MOI, multiplicity of infection; siRNA, small interfering RNA; SD, standard deviation; PK15, porcine kidney 15.