Mechanism Of Newcastle Disease Virus Inducing The Degradation Of Mitochondrial Antiviral Signaling Protein

Newcastle Disease (ND), caused by Newcastle Disease virus (NDV), can result in a variety of poultry deaths, which usually leads to severe losses in the poultry industry around the world. NDV is classified as a member of the paramyxoviridae, subfamily of paramyxovirinae. It possesses a single-stranded, negative-sense continuous RNA genome, which has a structure of 3’-NP-PMF-HN-L-5’.The RNA genome encodes six viral structural proteins respectively.During virus infection, multiple immune signaling pathways are triggered, acting in concert to mediate innate antiviral immunity and to initiate the inflammatory response against infection. The RIG-I-like receptor (RLR) family of pattern recognition receptors (PRRs) is a group of cytosolic RNA helicase proteins that can identify viral RNA as nonself via binding to pathogen associated molecular patter (PAMP) motifs within RNA ligands. This interaction then leads to triggering of an innate antiviral response within the infected cells through RLR induction of downstream effector molecules including MAVS, and induces antiviral and inflammatory gene expression. Furthermore, many viruses have been shown to prevent recognition of their viral RNA in the cytoplasm by the RLRs or target RLR pathways to escape from the immune response.To find the immune evasion mechanism of NDV on RLR signaling pathway, we detect expression of MAVS protein affected by infection of NDV in cells.1. MAVS was decreased in NDV infected cellsIn our study, HeLa or A549 cells were infected with NDV Herts/33, ZJ1, La Sota, and subsequently, the expression level of MAVS was analyzed by western-blot. The results reflected that MAVS was upregulated after NDV infect cells in early response, while NDV induced MAVS degradation in a delayed stage.It was demonstrated that RLR signaling pathway was strongly activated in early response,while it was almost completely destroyed latter.2. NDV induced MAVS degradation was not depended on PCBP2Data shows that PCBP2 was induced after viral infection, and its interaction with MAVS lead to proteasomal degradation of MAVS. It also observed lower susceptibility to infection with NDV in which PCBP2 expression was eliminated by RNAi.To verify whether the MAVS degradation induced by NDV was depended on PCBP2, HeLa or A549 cells were infected with NDV Herts/33, ZJ1, La Sota, and subsequently, the expression level of PCBP2 was analyzed by western-blot.The results showed that expression of PCBP2 was not upregulated in NDV infecting cells, it also not observed lower susceptibility to infection with NDV in which PCBP2 expression was eliminated by RNAi. Overexpression of PCBP2 did not lead to loss of endogenous MAVS. It was demonstrated that NDV induced MAVS degradation was not depended on PCBP2.3. NDV triggered proteasomal degradation of MAVSExperiments above have confirmed that NDV can degrade MAVS. Ubiquitin-proteasome system mediates most eukaryotic protein degradation as an important system.We choose MG132 as a proteasome inhibitor to verify whether the the degradation of MAVS was mediated by ubiquitin-proteasome system. Result showed that the MAVS loss induced by NDV was blocked by the proteasome inhibitor MG132, and the virus titer was reduced accordingly, indicating that NDV triggered proteasomal degradation of MAVS.4. The detection of MAVS degradation by NDV proteinsIn this research ZJ1 P/V/W eukarya expression plasmids were constructed to detect whether NDV proteins can degrade MAVS directly. We transfected ZJ1 P/V/W eukarya expression plasmids and Herts/33 P/M/NP eukarya expression plasmids which have been stored in our lab first, then the expression level of MAVS was detected. The results showed that there was no effect on the expression of MAVS when HA-P/W、Flag-P/NP/M are transfected. While the expression level of MAVS decreased in Flag-V transfected cells. In conclusion, the nonstructural protein V of NDV can degrade MAVS.In the current study, we confirmed that MAVS was decreased in NDV infected cells and NDV induced MAVS degradation was not depended on PCBP2 up-regulation. For the first time we propose that NDV V protein can degrade MAVS, which may provide a new thread to study NDV escapeing RLR signaling pathway.