Mechanism Of The Nucleolar Protein Lyar Promoting Influenzaa Virus Replication

Influenza A virus ribonucleoprotein(vRNP)complexes are responsible for transcription and replication of the viral genome in infected cells,which relies on the interactions of vRNP with host factors.Investigation of the interplay between vRNP and host proteins not only improves understanding of the molecular mechanism of viral transcription and replication but also discoveries antiviral targets from the host system,providing novel ideas for anti-flu drugs development.In this study,we have identified 80 host factors that copurified with vRNP using affinity purification followed by mass spectrometry.LYAR,a cell growth-regulating nucleolar protein,has been shown to be important for influenza A virus replication.Further studies demonstrate that LYAR interacts with vRNP,resulting in enhancing vRNP assembly,thereby facilitating viral RNA synthesis.In these processes,the nuclear localization together with S215 and S244 phosphorylation of LYAR are required,and LYAR phosphorylation is modulated by the virus activated protein kinase C(PKC).In the other hand,LYAR suppresses IFN-? production by targeting IRF3,negatively regulating antiviral innate immune responses,which leads to the enhanced virus replication.1.Identification of vRNP-interacting host proteinsReconstituted vRNPs of H1N1/PR8 virus were purified by using the Flag-PB1,followed by a LC-MS/MS to determine the binding partners and 80 host proteins were identified as the potential vRNP-interacting partners.For further study,we focused on the nucleolar proteins,and a siRNA-screen(LYAR,PPP1CA,HNRNPR,MCDRH and RPL19)was performed to investigate their effects on IAV replication and polymerase activity.LYAR displayed the strongest inhibitory effect on virus replication and polymerase activity when silenced.We therefore focused our investigation on the LYAR-vRNP interaction.2.LYAR interacts with vRNPCo-IP experiments showed that LYAR interacted with all the components of RNP when coexpressed.In addition,LYAR interacts with vRNP complex via its lysine rich C-terminal portion.The further study showed that LYAR interacted with all the RNP subunits in IAV infected cells,suggesting a real interaction between LYAR and RNP subunits during infection.Furthermore,when coexpressed,LYAR colocalized with PB1,PB2,and NP in the nucleoli,but colocalized with PA in the nucleoplasm.However,in virus infected cells,LYAR colocalized with NP in the nucleoplasm and cytoplasm,indicating that IAV infection triggers translocation of LYAR.These results suggest that LYAR may have a role in IAV transcription,replication,or vRNP nuclear export.3.LYAR facilitates viral RNA synthesisThe effect of LYAR on IAV replication was further examined.Results showed that the replication of H1N1 and H5N1 were significantly inhibited by LYAR knockdown or knockout mediated by siRNA or CRISPR/Cas9,indicating an important role of LYAR in IAV replication.We then explored the mechanisms of the LYAR-influenced IAV replication.We found that the synthesis of viral RNAs were significantly reduced or increased when LYAR silenced or overexpressed.Further study show that LYAR does not affect viral primary transcription,but it facilitates virus genome replication.Effects of LYAR on viral polymerase activity and vRNP assembly were further detected.The results indicate that LYAR facilitates virus genome replication by promoting vRNP assembly.4.Nuclear localization together with S215 and S244 phosphorylation of LYAR are important for IAV replicationThe further study show that nuclear localization together with S215 and S244 phosphorylation of LYAR are important for IAV replication.Deletion of NLSs and dephosphorylation of S215 and S244 significantly reduced the ability of LYAR to promote virus replication,but the phosphomimetic forms enhanced this ability,suggesting NLSs as well as S215 and S244 phosphorylation of LYAR are required for IAV replication.Additionally,the nuclear localization of LYAR is regulated by S215 and S244 phosphorylation.5.PKC regulates the nuclear localization of LYAR and IAV replicationPKC was predicated as a potential protein kinase of LYAR S215 by using NetPhos 3.1,which was verified in the further study.Given that phosphorylation of LYAR is important for its nuclear import and IAV replication,and S215 phosphorylation is regulated by PKC,we further examined the effect of PKC on the LYAR localization and IAV replication.Using the PKC activator(TPA)and inhibitor(BIM)in the experiments,we found that LYAR efficiently localized to the nucleoli after TPA treatment,while LYAR was distributed throughout the nucleus with some retained in the cytoplasm,demonstrating that the nuclear import of LYAR depends on its phosphorylation modulated by PKC.Further experiments were done to determine the regulation of PKC in IAV replication.The data showed that TPA promoted the RNA synthesis,viral polymerase activity,vRNP assembly,and virus replication,but the BIM showed an inhibitory effect.These results demonstrate that both LYAR efficient nuclear localization and IAV replication require LYAR phosphorylation regulated by PKC.Together with that IAV infection induces the phosphorylation of LYAR,we conclude that IAV infection activates PKC,resulting in phosphorylated LYAR,which in turn facilitates virus replication.6.Virus induced IFN-? enhances the expression of LYARIAV,Sev,and VSV infection or poly(I:C)stimulation leaded to a significant increase in the levels of LYAR mRNA and protein.In addition,the expression of LYAR was significantly increased in IFN-? treated A549 cells while did not significantly change in Sev stimulated IFN-I deficient Vero cells,which suggests that IFN-? increases LYAR expression,indicating a potential role of LYAR in antiviral innate immune responses.7.LYAR negatively regulates antiviral innate immune responses by targeting IRF3The study suggest that LYAR inhibits the production of IFN-?,ISGs and proinflammatory cytokines.Further studies showed that LYAR suppressed the activity of IFN-?,ISRE,and NF-?B promoter.Based on these findings,the effect of LYAR on RIG-I mediated IFN-? inducing pathway was analyzed,and we found that LYAR negatively regulates the FN-? by targeting IRF3.The mechanism of LYAR regulating FN-? via IRF3 was further investigated.The results showed that LYAR did not significantly affect phosphorylation and nuclear import of IRF3.However,LYAR interacts with IRF3 and impedes IRF3 binding onto ISRE,but does not affect the IRF3-CBP/P300 interaction.Therefore,LYAR suppresses FN-? production by inhibiting IRF3 DNA binding ability.Taken together,our studies have identified many potential vRNP-interacting host proteins,and reveal the mechanisms of how the nucleolar protein LYAR interacts with vRNP and promotes IAV replication,which not only improves current knowledge on the mechanisms of IAV transcription and replication but also uncovers LYAR new functions.