Molecular Mechanism Of BPIV3 Entry Into MDBK Cells

Bovine parainfluenza virus type 3(BPIV3)belong to genus Respirovirus and Paramyxoviridae family,which is one of the important pathogens of bovine respiratory disease(BRD).The mass epidemic of BPIV3 causes massive financial losses to the global cattle industry,there is no antiviral drug available for BPIV3 specific treatment.Viruses entry into the host cells is an important step in their life cycle and is also the golden age of antiviral therapy.Studying the molecular mechanism of viral entry of host cells can provide new potential targets for the development of antiviral drugs.However,the molecular mechanism of BPIV3 entry into cells is remain elusive.It has been reported that viruses can enter cells using host cell endocytosis,including clathrin-mediated endocytosis(CME),caveolae-mediated endocytosis(Cav ME),macropinocytosis,etc.We have demonstrated that BPIV3 can enter MDBK cells through clathrinmediated endocytosis and macropinocytosis pathways by chemical inhibitor treatment and RNA interference.Further studies revealed that the endosomal acidic environment and cathepsin L were also critical for the entry of BPIV3.Dynamin plays a key role in cell membrane fission during vesicle formation.It is required for clathrin-mediated endocytosis(CME)and caveolae mediated endocytosis(Cav ME)to occur.We found that dynamin is activated during BPIV3 entry and is involved in clathrin-mediated endocytosis.Cytoskeletal proteins(F-actin and microtubulin)can regulate vesicle formation and transport during endocytosis.The present study revealed that the entry process of BPIV3 was dependent on the rearrangement of F-actin,but microtubulin is not involved in this process.Regulation of dynamic in intracellular require the activation of corresponding receptors on the cell membrane surface.This is then transduced through a series of signalling pathways,ultimately leading to the rearrangement of cellular F-actin.It has been reported that sialic acid is a receptor for BPIV3 entry into host cells,but sialic acid receptors do not mediate the transduction of cellular signalling pathways.We hypothesize that there are other co-receptors involved in the signalling regulation of F-actin rearrangement during BPIV3 infection.Many viruses often use integrins and Receptor Tyrosine Kinase(RTK)to regulate the rearrangement of the F-actin.We detected BPIV3 entry after treatment of cells with receptor tyrosine kinase(RTK)inhibitors and found that BPIV3 entry was significantly inhibited by inhibition of tyrosine kinase(RTK)activity.In the RTK family,the epidermal growth factor receptor(EGFR)signalling pathway is used by many viruses to promote their entry into cells.We have shown that the EGFR receptor plays an important role in the rearrangement of F-actin by inhibiting EGFR activity during BPIV3 infection,by using pharmacological inhibitors and RNA interference,constructing mutants.Further studies revealed that when EGFR activity was inhibited,in addiimtion to BPIV3 entry being blocked,activation of the downstream PI3K-AKT and ERK1/2 and Rac1/Pak1 pathways was also significantly inhibited.we confirmed that BPIV3 enteres MDBK cells through the clathrin-mediated endocytosis(CME)and micropinocytosis.The occurrence of endocytosis is also dependent on the formation of a low p H environment in the endosome and the involvement of cathepsin L.We further confirm that the occurrence of the macropinocytosis pathway during BPIV3 entry into MDBK cells is dependent on the activation of the EGFR signaling pathway.In addition,PI3K-AKT and ERK1/2 、Pak1/Rac1 were activated in an EGFR-dependent manner during BPIV3 infection.In conclusion,this study illustrates the molecular mechanism of BPIV3 entry into MDBK cells.It provides a theoretical basis for the prevention and treatment of BPIV3 and a new target for the development of antiviral drugs.