HDAC6 Restricts Influenza A Virus Genome Transcription And Replication

Influenza A virus(IAV)belongs to a Genus in the Orthomyxoviridae family.As a negative-sense,single-stranded RNA virus,it is more subjected to mutation.RNA polymerase,nucleoprotein and viral genome constitute the ribonucleoprotein complex of Influenza A virus,which is the basic unit of transcription and replication of viral genome.Post-translational modifications play an important role in the life cycle of Influenza A virus.HDAC6,as a histone deacetylase,regulates the cytoskeleton of tubulin and mediates the antiviral immune response,or directly regulates viral proteins in the life cycle of various viruses.Thus,this study is based on the interaction between Influenza A virus and host protein HDAC6,found that acetylation modification of RNA polymerase complex PA protein plays an important role in polymerase activity and even viral genome transcription and replication,which provides reference for the the regulation of other viral RNA polymerases.1.HDAC6 interacts with Influenza A virus RNA polymerase complex proteinsInfluenza A viruses utilize hosts' materials and hijack host proteins to maintain their life cycle.Host protein HDAC6 inhibits the transport of virion via regulating the acetylation status of ?-tubulin,meanwhile viruses utilize HDAC6-mediated aggresome formation to promote their uncoating,during which process,M1 and NP interact with HDAC6.The nucleoprotein NP binding to viral RNA polymerase and viral genomic RNA forms the vRNP complex,therefore,through co-immunoprecipitation assay,GST pull-down and confocal immunofluorescent assay,we found that HDAC6 interacted with viral protein PA,PB1,PB2 and NP.Interestingly,HDAC6 could down-regulate PA protein level through proteasome degradation pathway,while had no effect on other viral proteins.Moreover,GST pull-down assay showed that HDAC6 binded to PA directly.Finally,we found that HDAC6 co-localized with PA in the cytoplasm by confocal immunofluorescence assay.Taken together,HDAC6 interacts with viral protein PA and regulates its protein level.2.HDAC6 deacetylates viral protein PA and affects its protein stabilityAs a histone deacetylase,HDAC6 is involved in the life cycle of various viruses.In this study,we explored the interaction mechanism between HDAC6 and PA based on the deacetylase activity of HDAC6.The level of PA protein was up-regulated in cells treated with HDAC6 specific inhibitor Tubacin or HDACs pan inhibitor TSA,However,HDAC6 but not HDAC6 catalytic activity deficient mutant(HDAC6-DM)could down-regulate PA protein level,indicating that down-regulation of PA by HDAC6 depends on its deacetylase activity.Importantly,we found that lysine 664 of PA protein was modified by acetylation and ubiquitination by mass spectrometry.In addition,in vitro deacetylation assay showed that HDAC6 deacetylated PA protein at lysine 664 in a deacetylase activity dependent manner.Moreover,the stability of PA protein is also regulated by acetylation status of lysine 664.In summary,HDAC6 modulates PA protein stability via deacetylating it at lysine 664.3.Overexpression of HDAC6 inhibits Influenza A virus genome transcription and replicationThree proteins PA,PB1 and PB2 constitute the Influenza A virus RN A polymerase complex,which mediates viral genome transcription and replication.Viral protein PA has endonuclease activity,which plays an important role in the formation of RNA polymerase trimer,and histone deacetylase HDAC6 can mediate PA protein deacetylation,and thus affect the protein stability of PA.In this study,immunoblotting,RT-PCR and real-time PCR were performed to detect virus replication in MDCK and A549 cells that were overexpressing HDAC6.The result showed that HDAC6 had an inhibitory effect on the transcription and replication of Influenza A virus.In addition,immunofluorescence assay and flow cytometry analysis showed that,the number of IAV/GFP(RFP)-HDAC6 double positive cells was significantly lower than IAV/GFP(RFP).Virus titer was also reduced upon overexpression of HDAC6.In summary,HDAC6 is able to inhibit viral proliferation by affecting the transcription and replication of Influenza A virus.4.Inhibition of HDAC6 promotes Influenza A virus genome transcription and replicationTo further confirm the role of HDAC6 in the life cycle of Influenza A virus,HDAC6 specific inhibitor Tubacin was used in MDCK and A549 cells to analyze its effect on virus amplification.Immunoblotting,RT-PCR and real-time PCR showed that Tubacin up-regulated the transcription and replication of Influenza A virus genome.Similarly,immunofluorescence and viral titer assay showed that Tubacin promotes the proliferation of Influenza A viruses.Subsequently,A549 cells were transfected with siHDAC6 to knock down HDAC6 followed by virus infection,it was found that viral genome transcription and replication were both up-regulated,and the virus titer was also increased,consistent with the inhibitor result.In summary,inhibition of HDAC6 deacetylase activity or silencing HDAC6 protein expression enhances transcription and replication of Influenza A virus.5.HDAC6 inhibits Influenza A virus RNA polymerase activity depending on its deacetylase activityThe histone deacetylase HDAC6 contains two independent deacetylase activity domains,which can mediate the deacetylation of ?-tubulin,heat shock protein 90,cortactin,etc.In addition to cellular proteins,we found that HDAC6 also can mediate the deacetylation of viral protein PA,affects its protein stability,and therefore inhibits viral transcription and replication.In this study,we found that overexpression of HDAC6,but not its catalytic activity deficient mutant(HDAC6-DM),can inhibit IAV RNA polymerase activity by mini-genome assay.Treatment with HDAC6 inhibitor Tubacin showed that IAV RNA polymerase activity was increased.Immunoblotting result also showed that overexpression of HDAC6 down-regulated IAV infection,while HDAC6-DM had no effect on virus.Taken together,inhibition of IAV RNA polymerase activity by HDAC6 is dependent on its deacetylase activity.