The Mechanism Of MERTK Promoting Replication Of Classical Swine Fever Virus

Viruses replicate within cells,and only after they enter host cells can they replicate and produce progeny virions.The virus entry into host cells requires the participation of receptors on host cell membrane,and these receptor proteins also determine the host range and tissue affinity of different viruses.Therefore,it is of great significance to search for the cellular receptor of virus and identify the entry mechanism of virus,to provide a new target for the design of antiviral strategies,to provide new insight into vaccine research,and contribute to the prevention and treatment of the disease.Classical swine fever,an acute and severe infectious disease in pig caused by classical swine fever virus?CSFV?,is prevalent in many countries and has caused serious economic losses to the world's pig industries.So far,two attachment receptors of heparin sulfate and laminin receptor were identified for CSFV,and both interact with E^rns of CSFV.However,E1 and E2 glycoproteins are sufficient to mediate CSFV entry,indicating that E^rns is not necessary for CSFV entry.Additionally,other studies have shown that E2 protein may interact with one cellular membrane protein to promote CSFV entry,but the specific cellular membrane protein is not clear.For screening new membrane proteins involved in CSFV entry,we found 12 candidate membrane proteins from two transcriptomics,and knockdown of four proteins,MERTK,TYRO3,CD97 and CD69,could significantly reduce CSFV protein expression by siRNA screening using a recombinant CSFV that contains a luciferase reporter to measure CSFV protein expression.Subsequently,we prioritized MERTK for the further research.Our results showed that overexpression of MERTK promotes CSFV replication.Both anti-MERTK polyclonal antibody and soluble MERTK^ED-His protein can decrease CSFV infection in a dose-dependent manner.Moreover,anti-MERTK polyclonal antibody also reduces the infection of genotype 2 CSFV.The Co-IP and surface plasma resonance assays demonstrated that MERTK interacts with CSFV E2 protein.The entry assay showed that MERTK facilitate CSFV entry into PK-15 cells.Additionally,LDC1267,an inhibitor of the tyrosine kinase of MERTK,reduce CSFV infection in a dose-dependent manner.Compared with control cells,the mRNA levels of IFN?in PK-15cells treated with LDC1267 and siMERTK were significantly upregulated after CSFV infection,suggesting CSFV dampens the innate immune through the activation of MERTK in PK-15 cells.The LDC1267 treatment has no effect on the CSFV entry,indicating that CSFV entry is independent of the kinase of MERTK.Interestingly,soluble MERTK^ED-His can block the infection of BVDV.In summary,our results demonstrated that the interaction of E2 and MERTK facilitates CSFV entry and the activation of the tyrosine kinase of the MERTK antagonizes the innate immune response in PK-15 cells.Moreover,MERTK play an important role in the infection of gene type 2 CSFV and BVDV.