Network of Interactions between the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus and Host Proteins
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Viruses, Antibodies, and Reagents
2.2. Construction of Recombinant Expression Plasmids
2.3. Y2H Screening
2.4. Functional Analysis of Interacting Proteins
2.5. Glutathione S-Transferase Pull-Down Assays
2.6. Co-Localization Analysis in Cells
2.7. Co-Immunoprecipitation Assays
2.8. The Effects of Signal Sequence Receptor Subunit 4 on APPV Infection
2.9. Western Blotting Assay
2.10. Quantitative Real-Time Polymerase Chain Reaction
2.11. Statistical Analysis
3. Results
3.1. Screening of the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus Interacting Proteins Using the Y2H System
3.2. Construction and Analysis of the Mut Domains of the E2 Protein–Cellular Protein Interaction Network
3.3. GO and KEGG Enrichment Analyses
3.4. Mut Domains of the E2 Protein Interact with CTSH and SSR4
3.5. Effects of SSR4 on the Replication of APPV
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Name | Forward Primer (5′-3′) | Reverse Primers (5′-3′) |
---|---|---|
pGBKT7/pEGFP-Mut | GAATTCATGCTCACCACCACCTGG | GGATCCCCTATTGGGCAGACAAGA |
pET-GST-Mut | GGATCCATGCTCACCACCTGG | GAATTCCCTATTGGGCAGACAAGA |
pCMV-tag4A-CTSH | GAATTCATGTGGGCCGTCCTGTC | CTCGAGCACCAGGGGGATCGGG |
pCMV-tag4A-SSR4 | GAATTCATGGCGGCGCTGG | CTCGAGGGCCTGGATGTGGCTC |
SSR4 (qRT-PCR) | ATCTCCACAGAGACCGTGTT | GCTGTAGGACTCCTCATCGAAG |
β-actin (qRT-PCR) | CACCATTGGCAATGAGCGGTTC | AGGTCTTTGCGGATGTCCACGT |
siSSR4-#1 | GAUCACGCCCUCGUACUAUtt | AUAGUACGAGGGCGUGAUCtt |
siSSR4-#2 | GGAAGGCUCAGAGAAAUAAtt | UUAWUUCUCUGAGCCUUCCtt |
siSSR4-#3 | GCAAGAACAGGGUCCAGAACAtt | UGUUCUGGACCCUGUUCUUGCtt |
Negative control (NC) | UUCUCCGAACGUGUCACGU | AAGAGGCUUGCACAGUGCA |
Gene | Protein | NCBI Accession | Function |
---|---|---|---|
SSR4 | Translocon-associated protein subunit delta | XM_003135482.5 | Regulates the retention of ER resident proteins |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | XM_021091114.1 | Modulates the organization and assembly of the cytoskeleton and innate immunity |
HINT1 | Histidine triad nucleotide-binding protein 1 | XM_005661623.3 | Involved in phosphorylation of ribonucleotide |
CTSH | Cathepsin H | XM_017021951.2 | Involved in proteolysis associated with protein catabolic process |
STIP1 | Stress-induced phosphoprotein 1 | XM_003353794.4 | Co-chaperone that binds to heat shock proteins 70 and 90 |
POP7 | Ribonuclease P protein subunit p20 | XM_013995472.2 | Involved in rRNA processing, tRNA 5′-leader removal, and tRNA processing |
PHGDH | D-3-phosphoglycerate dehydrogenase | XM_011541226.3 | Involved in the early steps of L-serine synthesis in animal cells |
RPS25 | 40S ribosomal protein S25 | NM_001028.3 | Component of the small ribosomal subunit |
CKB | Creatine kinase B-type | XM_021081502.1 | Reversibly catalyzes the transfer of phosphate between ATP and various phosphagens |
AKT1 | RAC-alpha serine/threonine-protein kinase | XM_021081500.1 | Protein serine/threonine kinase activity |
NME2 | Nucleoside diphosphate kinase B | NM_001044610.2 | Involved in the synthesis of nucleoside triphosphates other than ATP |
RPS26 | Small ribosomal subunit protein eS26 | NM_001097481.2 | Structural constituent of the ribosome |
S100A2 | S100 calcium-binding protein A2 | XM_001929556.5 | Calcium ion binding |
ACTG1 | Actin gamma 1 | XM_003357928.4 | Involved in various types of cell motility and maintenance of the cytoskeleton |
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Yang, Y.; Jiang, G.; He, W.; Tian, X.; Zheng, H.; Xiang, B.; Sun, Y. Network of Interactions between the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus and Host Proteins. Genes 2024, 15, 991. https://doi.org/10.3390/genes15080991
Yang Y, Jiang G, He W, Tian X, Zheng H, Xiang B, Sun Y. Network of Interactions between the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus and Host Proteins. Genes. 2024; 15(8):991. https://doi.org/10.3390/genes15080991
Chicago/Turabian StyleYang, Yuai, Guangfei Jiang, Weiqi He, Xin Tian, Huanli Zheng, Bin Xiang, and Yongke Sun. 2024. "Network of Interactions between the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus and Host Proteins" Genes 15, no. 8: 991. https://doi.org/10.3390/genes15080991
APA StyleYang, Y., Jiang, G., He, W., Tian, X., Zheng, H., Xiang, B., & Sun, Y. (2024). Network of Interactions between the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus and Host Proteins. Genes, 15(8), 991. https://doi.org/10.3390/genes15080991