| Porcine transmissible gastroenteritis is caused by porcine transmissible gastroenteritis Virus which can cause piglets vomiting, diarrhea, dehydration, intestinal villi atrophy, high mortality and high contagious disease characteristics. Less than two week old piglets infection rate reached 100%, adult pigs carry the virus but not sick, but slow growth,continuous detoxification, low immunity, secondary infection of other pathogens, resulting in huge economic losses in pig industry of China. TGEV mainly infects the intestinal epithelial cells, but the mechanism of its invasion is not clear, actin cytoskeleton plays an important role in mediating the virus invasion process, in the present study, we found that early TGEV infection can cause the rearrangement of the actin cytoskeleton in intestinal epithelial cells, but the specific regulation mechanism is not clear. The first step in virus invasion is the receptor binding on the surface of the cell membrane, a growing number of stadies have found that virus invading host cells may require the collaboration between multiple receptors. In this study, we explore the mechanism of TGEV regulation of actin cytoskeleton rearrangement and new receptor of TGEV invasion by using porcine jejunum epithelial cells (IPEC-J2) as the infection model. To provide a theoretical basis for the prevention and control of TGEV, this research content is divided into the following four parts:1. Complete genomic sequence of the coronavirus transmissible gastroenteritis virus SHXB.TGEV SHXB was isolated in Shanghai, China, in order to further understand the evolution of genetic information, the complete genome of strain SHXB was sequenced, and its sequence was compared those of other TGEV strains in the GenBank database. The comparison showed that there were no insertions or deletions in the 5’and 3’ non-translated regions, in the nonstructural genes ORF1, ORF3, and ORF7, or in the genes encoding the structural proteins envelope (E), membrane (M) and nucleoprotein (N). A phenomenon in common with other strains was that nucleotide (nt) 655 of the spike (S) gene was G, and acommon change in nt 1753 of the S gene was a T-to-G mutation that caused a serine-to-alanine mutation at amino acid 585, which is in the region of the main major antigenic sites A and B of the TGEV S protein. A 6-nt deletion was also found at nt 1123-1128 in all Purdue strains except the strain Virulent Purdue. Phylogenetic analysis showed that TGEV SHXB was closely related to the Purdue strains and shared a common ancestor with the Miller strains as well as strain PRCV-ISU-1.2. TGEV caused the rearrangement of the actin cytoskeleton of IPEC-J2 cells in the early infection.Cell shape, movement, phagocytosis, cell organelles, and intercellular communication require the involvement of the actin cytoskeleton. There have been many reports about viral infection caused the rearrangement of actin, actin mediated virus invasion and plays an important role in the process of internalization. This study had found that TGEV infection(MOI=2) caused the rearrangement of actin by fluorescence microscopy, cortical layer actin rearrangement, the changes of cortical layer actin caused the formation of filopodia,lamellar pseudopodia and lamellipodia in the cell membrane. Electron microscopy revealed that the actin was located around the virus, confocal microscopy revealed that the virus could be co-located with the actin filament, actin cytoskeleton inhibitors significantly inhibited the invasion of TGEV (p < 0.01). These results show that TGEV causes actin to gather around the cell membrane, actin is involved in the TGEV entry process. Cofilin had a central role in controlling actin dynamics by regulating actin polymerization and depolymerization through its severing activity, the level of p-cofilin increased in the early infection of TGEV. Over-expression, interference and site directed mutagenesis cofilin significantly inhibited the invasion of TGEV (p < 0.01). Cofilin is proved to regulate the actin cytoskeleton early in TGEV infection.3. Study on the mechanism of TGEV infection causes the rearrangement of actin cytoskeleton in IPEC-2 cells.TGEV early infection stimulated cortical actin cytoskeleton rearrangement, cell stress fibers disappeared, actin was involved in the TGEV entry process, actin depolymerizing factor cofilin played an important role in this process. We further explored the regulation mechanism of cofilin early in TGEV infection, Point mutations in the Rho-GTPases family significantly inhibited the invasion of TGEV (p < 0.01), PI3K was participated in the early regulation of cofilin. The epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) belongs to the RTK family, TGEV infection induced the activation of EGFR, TGEV was co-localized with EGFR, EGFR specific inhibitor AG1478 significantly inhibited TGEV entry (p < 0.01) and the activation of downstream signaling pathways.These results prove that EGFR is involved in TGEV entry and cofilin phosphorylation.Lipid rafts are mainly composed of sphingolipid and cholesterol, lipid rafts inhibitor removal of cell membrane lipid rafts could significantly inhibit the adhesion and invasion of TGEV (p < 0.01), lipid rafts clustered in TGEV infected cells and gathered around EGFR,removal of lipid rafts could significantly inhibit the activation of EGFR and downstream signaling pathways early in TGEV infection. These results indicate that lipid rafts are involved in the regulation of actin cytoskeleton by cofilin. TGEV stimulates the phosphorylation of cofilin and the polymerization of actin through EGFR-PI3K-Racl/Cdc42-PAK-LIMK signaling pathway to promote the invasion of TGEV.4. Study on the mechanism of EGFR mediates TGEV entry.TGEV stimulates the phosphorylation of cofilin and the polymerization of actin through EGFR-PI3K-Racl/Cdc42-PAK-LIMK signaling pathway, APN is a known receptor of TGEV. Further studies found that interference EGFR and APN at the same time could more significantly inhibit the invasion of TGEV (p < 0.01), EGFR receptor binding domain 1 protein can significantly inhibit the invasion of TGEV(p < 0.05), EGFR extracellular receptor binding domain 1 could interact with TGEV spike protein directly, TGEV early infection could promote APN and EGFR clustering, PI3K/AKT and ERK1/2 signaling pathways were involved in the internalization of TGEV, APN receptor shRNAs could significantly inhibit the activation of TGEV on EGFR, APN was able to stimulate EGFR and downstream signaling pathways. Both clathrin and caveolin targeting shRNAs significantly inhibited TGEV entry (p < 0.01), it indicated that TGEV entry via clathrin and caveolin mediated endocytosis in IPEC-J2 cells. In normal IPEC-J2 cells, most of EGFR located at the cell membrane surface, TGEV infection promoted EGFR internalization,clathrin targeting shRNAs significantly inhibited EGFR internalization, TGEV binds with EGFR and internalizes through clathrin-mediated endocytosis pathway. These results show that EGFR is another receptor of TGEV, and it plays a synergistic role with APN. |
PDF Full Text Request