The Mechanism Studies Of The Epidermal Growth Factor(EGFR) Signaling Pathway And Syndecan 4 In The Entry Process Of Porcine Reproductive And Respiratory Syndrome Virus

The porcine reproductive and respiratory syndrome virus, belonging to the family of Arteriviridae, genera of arteritisvirus, is an enveloped and positive-stranded RNA virus.PRRSV infection causes acute respiratory symptoms in young piglets and reproductive,failure in pregnant sows, which leads to considerable economic losses to the swine industry worldwide. Previous studies have been focused on the exploration of PRRSV infection and host immune response mechanism, however, the mechanism of adsorption and entry of PRRSV is not clear. This study focuses on the signaling pathway and its molecular mechanism during PRRSV entry, also, the attachment receptor of PRRSV is explored. The main contents of this thesis are the following:1. The role of protein tyrosine kinases in porcine respiratory and reproductive syndrome virusProtein tyrosine kinases mediate many inside and outside the cell signal transduction and processing. In the process of tunorigenesis, they played a key role in regulating cell growth and differentiation. We studied the role of protein tyrosine kinase in PRRSV entry to MARC-145 cells. The results showed that Src and EGFR were activated during PRRSV entry to cells, and the specific inhibitors Genistein, PP2, SU6656 and AG 1478 cells significantly inhibited PRRSV entry. It suggested that PRRSV utilized tyrosine kinase activity to facilitate virus entry to MARC-145 cells. Also, Src interacts with EGFR in MARC-145 cells.We also focus on the effect of EGFR on PRRSV entry to MARC-145 cells. The results showed that overexpression of EGFR or activation of EGFR promoted PRRSV entry to the cells, whereas reducing the expression of EGFR or inhibiting the activity of EGFR markedly decreased PRRSV entry. These results suggest that PRRSV activated Src and EGFR and related signaling molecules to promote it entry to the MARC - 145 cells.2. PRRSV entry activates the EGFR-PI3K/AKT-LIMK-cofilin signaling pathwayWe explored the signaling molecules that were activated during PRRSV entry to verify which signaling pathway is crucial for PRRSV entry. The results showed that the entry of PRRSV could induce the activation of PI3K, AKT, LIMK1 and cofilin. The signaling pathway EGFR-PI3K-AKT-ILIMK1-cofilin is essential for PRRSV entry. The activation of signaling pathway regulated the cytoskeleton to mediate the virus entry. Blocking EGFR by AG1478 attenuated the activation of PI3K, AKT, LIMK1, cofilin induced by PRRSV entry.The PI3K inhibitor LY294002 also decreased the activation of AKT, LIMK1, and cofilin.Moreover, ROCK inhibitor Y27632 disrupted the activation of LIMK1 and cofilin.Inhibitors to PI3K, AKT and ROCK, significantly inhibited the virus entry. Also,knockdown of cofilin significantly reduced PRRSV entry. Furthermore, over-expression of the constitutively active form of cofilin partially rescued the virus entry inhibited by deletion of cofilin in MARC-145 cells. In conclusion, our results reveal that PRRSV triggers the activation of EGFR-PI3K-AKT-LIMK1-cofilin signaling pathway to facilitate its entry into cells.3. Syndecan 4 involves in PRRSV attachment and entry to MARC-145 cellsIn our study, we explored the role of syndecan 4 in PRRSV attachment and entry to MARC-145 cells. We found knockdown of syndecan 4 significantly impaired PRRSV attachment, whereas syndecan 4 overexpression enhanced PRRSV attachment. Moreover,the overexpression of syndecan 4 increased PRRSV entry. Our results showed that syndecan 4 interacts with EGFR in MARC-145 cells. Disruption of the interaction reduced PRRSV entry while had no effect on virus binding. Furthermore, the immunofluorescence analysis showed that FGFR specific inhibitor AG 1478 and the peptide SSTN87-131 inhibited the entry of syndecan 4 accompanied with PRRSV entry. We proposed that upon PRRSV attached to syndecan 4, EGFR coupled with syndecan 4 was activated and further initiated the signaling pathways involved in virus entry.4. The signaling pathway mediated by syndecan 4 during PRRSV entryTo further understand the role of syndecan 4 in PRRSV entry, we studied the signaling mediated by syndecan 4 during PRRSV entry. The western blot analysis showed that knockdown of syndecan 4 significantly impaired the activation of FAK , Src and EGFR induced during PRRSV entry, Moreover, we found Annexin A2, a calcium binding protein,involved in PRRSV entry. The results showed that Annexin A2 was activated during PRRSV entry. Also, knockdown of Annexin A2 significantly inhibits PRRSV entry into the cells, and the overexpression of Annexin A2 promotes the virus into the cells. Furthermore,FAK specific inhibitor PF573228, Src inhibitor PP2 and EGFR inhibitor AG1478 can significantly inhibit the activation of Annexin A2 induced during PRRSV entry. Also,knockdown of Annexin A2 decreased the phosphorylation of EGFR, Src and cofilin induced during PRRSV entry. We suggested that Annexin A2 mediated PRRSV entry by regulating the activity of EGFR and Src, and by remodeling the cytoskeleton to promote virus into the cells. These results suggested that syndecan 4-FAK/SRC/EGFR-Annexin A2-cofilin signaling pathway is essential for PRRSV entry to MARC-145 cells.