Interaction Of FABP7 And MSI1 With Newcastle Disease Virus V Protein And Its Antiviral Mechanism

Newcastle Disease(ND)has affected the poultry industry around the world as a serious infectious disease since it was first reported in 1926.On the World Organization for Animal Health's list of notifiable diseases.Newcastle Disease Virus(NDV)is a single stranded RNA Virus with a capsule.According to the latest virus classification,it belongs to the Orthoavulavirus genus of Paramyxoviridae and Avulavirinae subfamily.As other paramyxoviruses,NDV encodes nonstructural proteins that are translated into V and W by RNA editing via inserting one or two additional guanine(G)at the conserved editing site of the P gene.The P,V,and W proteins contain a common amino terminal,while the carboxyl terminal is different.The length of V protein expressed by NDV was 239 amino acids and the molecular weight was 36 k Da.The recombinant NDV which cannot express V protein has low pathogenicity and increased sensitivity to exogenous interferon.The NDV Clone-30 mutant without the V protein was unable to reproduce in SPF chicken eggs,suggesting that the V protein plays an important role in viral replication.Studies have shown that the expression of V protein can block the activation of interference response through its carbon terminal domain,thus helping NDV to replicate successfully in host cells.Previous studies have shown that in interferon-deficient cells,the deletion of the carbon terminal domain of V protein reduces the replication level of RNDV,indicating that we have not fully understood the function of V protein.(1)In order to further study the function of V protein,some proteins interacting with V protein were screened by yeast two-hybrid technology in the previous work in our laboratory.FABP7(fatty acid binding protein 7)and MSI1(Musashi RNA binding protein 1)were selected to construct eukaryotic expression vectors for FABP7 and MSI1,respectively.The interaction between FABP7 and V or MSI1 with V was verified by Co-IP assay and immunofluorescence co-localization assay.The results showed that FABP7 and MSI1 could interact with NDV V protein.(2)We further studied the effect of FABP7 on the virus.First,we verified the expression of FABP7 vector.Then,FABP7 was the overexpressed or knockdown with si RNA in DF-1cells,and detected the content of the virus by Cell plaque test and Western Blot.The results showed that the overexpression of FABP7 in DF-1 cells could inhibit the replication of the virus.Meanwhile,the knockdown of endogenous expression of FABP7 with si RNA showed the opposite result.Further results showed that FABP7 overexpression strongly triggered the phosphorylation of IRF3 and increased the phosphorylation levels of STAT1 and TBK1.We speculated that FABP7 may promote the phosphorylation of IRF3 by up-regulating the phosphorylation level of TBK1,which is transferred into the nucleus,and enhances the expression of interferon,activates the downstream signaling pathwa,promotesthe phosphorylation of STAT1,and up-regulates the expression of interferon-related stimulators,thereby inhibiting viral replication.(3)We studied the effect of MSI1 on NDV.First,we verified the expression of MSI1 vector in DF-1 cells.MSI1 was then expressed in DF-1 cells,and detected the content of the virus by Q-PCR,Cell plaque test and Western Blot.Q-PCR and Western Blot detection showed no significant difference in the levels of viral m RNA and protein in cells.However,the number of virions in the supernatant was detected by cell plaque assay,and found that the overexpression of MSI1 reduced quantity of virions in the supernatant.Subsequently,we found that overexpression of MSI1 inhibited cell apoptosis,suggesting that MSI1 may affect the release of virions by inhibiting cell apoptosis caused by NDV infection.In conclusion,the V protein of NDV may affect viral replication by interacting with MSI1 and FABP7.