The Interaction Between Neddylation Modification And Pathogenicity Of Influenza A Virus

Influenza A virus(IAV)belongs to the Orthomyxoviridae family.The pathogenicity of IAV is determined by both viral and host factors.Previous studies showed that lung injury and cytokine storm were important reasons for IAV infection caused morbidity and mortality in animals and humans.Neddylation modification plays an important role in regulating virus infection.However,the relationship between IAV and Neddylation pathway is not clear.In order to uncover the interactions,the experiments were carried out in vivo and ex vivo.The main contents of this study including followings:1.The pathogenicity of IAV is related to pro-inflammatory responses,and neddylation modification plays an important role in regulating inflammatory responses.In order to select an IAV strain showing low pathogenicity but strong inflammatory responses,SPF chickens and BALB/c mice were experimentally infected with 4 strains of H9N2 subtype from different hosts.Pathogenicity of H9N2 strains in SPF chickens showed that all these four H9N2 strains replicated well in respiratory tract and induced inflammatory responses in the trachea and lungs.FACS analysis showed that percentage of CD8+ T cells slightly increased to 0.7%from 0.1%after infection with the A/swine/Jiangsu/C1/08(H9N2)(H9C1)isolate.Pathogenicity of H9C1 in mice showed that H9C1 replicated well in lungs and the expression levels of cytokines,IL-1?,IL-6,TNF-?,MCP-1 and MIP-1,increased significantly,indicating that H9C1 strain caused a strong inflammatory response.Taken together,H9C1 strain was selected as a model strain for further studies.2.The neddylation pathway belongs to post-translational modifications and plays important roles in regulating viral infection and replication.To address the relationship of IAV with the neddylation modification pathway,A549 cells were infected with H9C1 and pandemic H1N1 2009(pdmH1N1)(maCa04)and showed that IAV infection at an early stage(4?6 h)could elevate the level of Neddylated-Cullin-1 by 1.6?2.1 fold along with an apparent increase in pro-inflammatory cytokines(IL-1?,IL-6 and TNF-?).These suggest that IAV infection activates the neddylation modification pathway to promote virus infection and thus enhance the expression of pro-inflammatory cytokines leading to an increase in the pathogenesis.3.The treatment of Nedd8 E1 activating enzyme(NAE1)-specific inhibitor,MLN4924,is able to interfere with Nedd8 conjugation and NF-?B activity.To determine the effect of neddylation modification on IAV replication and pathogenicity,A549 cells were treated with MLN4924 and followed by infection of several subtypes of IAVs,including classical H1N1(PR8),H9C1,and maCa04 viruses.We found that blocking the neddylation pathway by MLN4924 was able to tremendously reduce IAV replication and inflammatory responses.To evaluate the antiviral activity of MLN4924 in vivo,BALB/c mice were intranasally inoculated with 200 ?g MLN4924 followed by infection of maCa04(104 TCID50),H9C1(105 TCID50)or PR8(5×104 TCID50).It showed that MLN4924 treatment not only remarkably decreased virus titers as well as pro-inflammatory cytokines(IL-1?,IL-6 and TNF-?)in the lungs of mice infected by low virulent strain H9C1 and high virulent strains maCa04 and PR8,but also saved 20%of mice infected by maCa04 and PR8.These findings suggest that MLN4924 could be potentially developed as a novel antiviral therapy for IAV infection.4.In order to characterize viral proteins interaction with key factors of neddylation modification pathway,cell lines that stably knock down Cullin-1 were generated by using lentivirus-mediated expression of shRNA.It showed that virus titters were obviously increased in Cullin-1 knockdown cells,indicating that Cullin-RING E3 ligase(CRL1)negatively regulates IAV infection.Next,Co-Immunoprecipitation was performed and showed that Cullin-1 interacted with NS1,but not with PB2,PB1,PA or NP.It also showed that Cullin-1 suppressed NS1 expression via mediating NS1 protein degradation.Moreover,RNA binding domain of NS1 is required for its association with cullin-1.Thus it is likely that NS1 could hijack the CRL1 complex and eliminate CRL-1-mediated degradation of viral proteins,the mechanism of which is of great interest for further studies.