The Studies Of The Signaling Molecules Involved In The Entry Process Of Porcine Reproductive And Respiratory Syndrome Virus

The porcine reproductive and respiratory syndrome virus,an enveloped and positive-stranded RNA virus,belongs to the family of Arteriviridae,order of Nidovirales.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.Although great efforts have been made to explore the pathogenesis of PRRSV,mainly focused on host immunity response,the factors that affect the different stages of PRRSV infection,such as virus entry,replication in host cells,virus assembly and release,are still poorly understood.This study is mainly focused on the signaling molecules and the molecular mechanism that is involved in PRRSV entry.The main contents of this thesis are the following:1 PRRSV entry activate the FAK-PI3K-AKT-Rac1 signaling pathwayWe tested the signaling molecules that can be activated during PRRSV entry to verify which signaling pathway is crucial for PRRSV entry.The results showed that the entry of PRRSV?JXA1?could induce the activation of FAK,PI3K,AKT and Racl signaling molecules,and the remodeling of actin cytoskeleton was shown.The signaling pathway FAK-PI3K-AKT-Racl is essential for PRRSV entry.Blocking FAK by PF573228 attenuates the activation of PI3K,AKT,Rac1 and the cytoskeleton remodeling induced by virus entry.The PI3K inhibitor LY294002 can also decrease the activation of AKT and Rac1.Inhibitors to FAK,PI3K,AKT and Racl can significantly inhibits the virus entry.Over-expression of constitutively active form of Racl can reverse the virus entry inhibition effect in AKT inhibitor treated MARC-145 cells.In conclusion,our results reveal that PRRSV triggers the activation of FAK-PI3K-AKT-Rac1 signaling pathway to facilitate its entry into cells.2 Integrin av subunit is involved in the activation of FAK-PI3K-AKT-Racl signling pathway induced by PRRSV entryWe further explored the functional role of integrins in PRRSV entry.We found that PRRSV associates with integrin av subunit but not with?1 subunit.PRRSV redistributes integrin av subunit to the plasma membrane during its entry.Additionally,blocking integrin av subunit by neutralization antibody or shRNA significantly attenuates both PRRSV internalization and the signaling transduction.Meanwhile,PI3K activator could reverse the virus entry inhibition effect in integrin av subunit knockdown MARC-145 cells.Furthermore,the integrin activator,manganese,can promote the activation of FAK-PI3K-AKT-Rac1 signaling pathway and enhance PRRSV entry.PRRSV entry induced the movement of integrin av subunit to lipid raft and the lipid raft is critical for integrin signaling transduction.In conclusion,our results reveal that PRRSV entry into cells mimics the outside-in activation process of integrin to facilitate PRRSV entry into MARC-145 cells.3 Cellular ROS and Ca²⁺ is crucial for signaling transduction and PRRSV entryWe studied the role of cellular ROS and Ca²⁺ in PRRSV entry.We found that PRRSV induced release of cellular ROS during its entry process.The cellular ROS is important for PRRSV entry,and reduce the cellular ROS by antioxidant NAC can significantly inhibit PRRSV entry.Furthermore,the NAC can also inhibit the activation of FAK-PI3K-AKT-Rac1 signaling pathway.PRRSV may induce cellular ROS release through TLR4.The specific inhibitor of TLR4,TAK242,can significantly reduce the intracellular ROS release.The TAK242 can also inhibit both PRRSV entry and the activation of FAK-PI3K-AKT-Racl signaling pathway.Besides,BAPTA-AM can also inhibit the PRRSV entry and the signaling pathway.In conclusion,our results showed that both cellular ROS and Ca²⁺ is crucial for the signaling transduction and PRRSV entry.4 PRRSV might bind to integrin through the RHD motif on GP3 proteinThe ligands bind to integrin through the specific motifs.According to the motifs that had been reported,we found two integrin binding motifs on PRRSV structural protein.We found RHD and LDV motifs on GP3 and GP2a respectively.These small peptides were chemically synthesized.Our results showed that RHD peptide can inhibit both the entry of PRRSV and attenuate the activation of FAK-PI3K-AKT-Rac1 pathway induced by PRRSV entry.Besides the RHD peptide did not inhibit the attachment of PRRSV.In conclusion,our results showed that PRRSV might bind to integrin through the RHD motif on GP3 protein.