| M1 is proposed to play multiple biologically important roles in the life cycle of IAV.Especially during virus budding and assembly,which largely relies on host factors by interaction.Identifying novel host proteins that interact with M1.Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development.In this study,we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of AIV(H5N6)through immunoprecipitation(IP)/mass spectrometry.Among them,PSMD12,a 26 S proteasome regulatory subunit,was shown to interact with influenza M1,acting as a positive host factor in AIV replication in avian DF1 cells.Mechanistically,PSMD12 mediates K63-linked ubiquitination of M1 at the K102 site,and thus positively regulates influenza virus proliferation,and an M1-K102 mutation affected the M1-M2 VLP formation.Furthermore,we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses,and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology.Additionally,we confirm the reduced virulence of JX(H5N6)and PR8(H1N1)viruses carrying the M1-K102 site mutation in mice.The main contents are as follows:1.Identification of host factors that interact with M1Fifteen of the 19 potential M1-interacting host proteins were successfully cloned into the PCAGGS vector,which effect of regulatory AIV proliferation was verified by overexpressing.Finally,we validated 3 positively regulated genes(greater than 2-fold increase in number of viral plaques),respectively is GAPDH,HIST1H2B5 L and PSMD12.And 5 negatively regulated genes(less than 0.5-fold increase in number of viral plaques),respectively is CALM2,DNAJC10,HSP70,HSPA8 and MYO1 D.Among them,the overexpression of PSMD12 promoted the replication of IAV is significantly.2.PSMD12 regulates the proliferation of H5N6To further verify the regulatory effect of PSMD12 on influenza virus,we designed three small interfering RNA(si RNAs)to target PSMD12 in avian DF1 cells.The results showed that knock down PSMD12 inhibited the proliferation of H5N6 virus.To evaluate the effect of knockdown PSMD12 on the proliferation of avian influenza viruses of H5N6 in human cells,three sh RNAs were designed to target PSMD12 in human A549 cells.Similarly,the results showed that the impaired expression of PSMD12 significantly decreased viral titer.These results suggest that both avian and human PSMD12 have a positive regulatory effect on the AIV of H5N6.3.Interaction between PSMD12 and M1The results of Co-IP experiments indicated that PSMD12 of avian/human source could interact with M1.And co-localization of PSMD12 with M1 was demonstrated in AIV(H5N6)-infected DF1/A549 cells,suggesting a genuine interaction of PSMD12 with M1 during virus infection.4.PSMD12 promotes ubiquitination of M1It was verified that M1 could be modified by ubiquitination.Interestingly,ubiquitination of M1 was enhanced by PSMD12 overexpression.The PSMD12-mediated M1 ubiquitination modification was further classified,and it was found that overexpression of PSMD12 obviously increased K63-linked,but not K6-,K11-,K29-,K33-,or K48-linked ubiquitination of M1.5.M1 K102 is crucial for PSMD12-mediated ubiquitination of K63Using BDM-PUB and UBPRED in this study predicted that there are nine amino acid site is most likely to undergo ubiquitination modification in M1 of H5N6.Further experiments were carried out to verify one by one,and we found that the K102 site of M1 is very important for PSMD12-mediated ubiquitination.The K102 site of M1 was also found to be highly conserved by comparing other subtypes of influenza viruses.6.M1 K102 site was associated with the proliferation of H5N6 virus promoted by PSMD12The M1 K102 R mutants of H5N6 and PR8(H1N1)viruses were successfully constructed by reverse genetics.PSMD12 overexpression promoted H5N6 virus proliferation,whereas PSMD12 knock down inhibited the proliferation of H5N6 on DF1 cells.However,neither overexpression nor knockdown of PSMD12 could significantly affect the proliferation of H5N6-M1-K102 R on DF1 cells.These findings provided further evidence that the mechanism of PSMD12 positively regulating the proliferation of H5N6 virus was related to the K102 site of M1.7.Mutations at M1 K102 inhibit viral proliferation in vitroThe proliferation of wild-type H5N6-M1 or PR8-M1 and the corresponding mutant strains H5N6-M1-K102 R or PR8-M1-K102 R were compared on different cell lines,and it was found that the proliferation ability of the virus after the M1 K102 site mutation decline.Interestingly,although viral yield in the supernatant was reduced after the M1K102 site mutation,viral protein was increased in the interior of the infected cells.It is suggested that the budding(release)of the virus may be inhibited after the mutation of the M1 K102 site of AIV(H5N6)or PR8(H1N1).8.M1 K102 mutation or knockdown PSMD12 restricts virus releaseCo-transfection of M1 and M2 could form release-type M1-M2 VLPs,and the ability to form M1-M2 VLPs decreased when M1-K102 R was co-transfected with M2.Further,at the virus level,it was observed by transmission electron microscopy that the budding of H5N6-M1-K102 R virus was inhibited.In addition,on the host side,knockdown PSMD12 also inhibited the budding of H5N6 virus on CEF cells.9.Mutations at M1 K102 site affected the Pathogenicity of AIV in miceThe pathogenicity of the M1 K102 site mutation on H5N6 and PR8 viruses in mice was further explored.First,the median lethal dose MLD50 of H5N6-M1 and PR8-M1 was determined by the infection experiment in mice.After adjusting the same infection dose of wild virus and mutant virus,the body weight changes,survival curves,histopathological sections,and tissue viral loads of infected mice were evaluated 0-14 days after infection.Conclusions: Mutation at the K102 site of M1 reduces the pathogenic and lethal ability of AIV(H5N6)and PR8(H1N1)in mice. |
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