Molecular Mechanism Exploration Of ABTB1 In Regulating The Replication Of Influenza Viruses

Influenza A virus(IAV)is an important epidemic pathogen and a kind of enveloped RNA virus with segmented genome.It causes acute respiratory infections,which seriously threatens the global public health security and generates huge economic losses.The invasion process of IAV into host cells depends on many host factors.Discovery and identification of host factors regulating the replication of IAV could provide a reference for the development of anti-IAV drugs.Ankyrin repeat and BTB/POZ domain-containing protein 1(ABTB1)is screened by small interfering RNA(si RNA)library in our laboratory as a host factor which may affect the replication of IAV.To explore the regulation effect of ABTB1 on the replication of IAV,si RNA interference,CRISPR/Cas9 knockout cell lines and p QCXIN overexpression cell lines were used in this study to explore the influence of ABTB1 on the titer of IAV.We found that downregulation of ABTB1 by si RNA interference or knockout of ABTB1 by CRISPR/Cas9 system significantly inhibited the replication of IAV,while stable overexpression of ABTB1 largely facilitated the replication of IAV,indicating that ABTB1 positively regulated the replication of IAV.Confocal observation showed that downregulation of ABTB1 by si RNA interference,knockout of ABTB1 and stable overexpression of ABTB1 affected the early stage of life cycle of IAV.Further investigation indicated that ABTB1 did not affect the binding,endocytosis,fusion and uncoating stages but positively regulated the nuclear import of vRNP.The nuclear import of vRNP of IAV was mainly mediated by NP protein.We found that ABTB1 did not interact with NP directly.Therefore,ABTB1 regulated the nuclear import of vRNP by means of an indirect manner.Immunoprecipitation and mass spectrometry assays confirmed that Tripartite motif-containing protein 4(TRIM4)was a host factor interacting with ABTB1,and in contrast with ABTB1,overexpression of TRIM4 significantly inhibited the replication IAV,while downregulation of TRIM4 by si RNA interference or knockout of TRIM4 largely promoted the replication of IAV.TRIM4 negatively regulated the nuclear import of vRNP of IAV by interacting with NP and degrading NP through K48-related ubiquitin proteasome system,which indicated that TRIM4 negatively regulated the nuclear import of vRNP by targeting and degrading NP protein of IAV.Finally,we explored the relationship between ABTB1 and TRIM4.We found that ABTB1 could degrade TRIM4 protein while TRIM4 expression elevated the expression of ABTB1.ABTB1 degraded TRIM4 through proteasome pathway and rescued the degradation of NP or the virus titer caused by the inhibition of TRIM4.According to mass spectrometry,Elongation factor 1-alpha 2(e EF1A2)interacted with ABTB1.It was reported that Sorting nexin-16(SNX16)prevented the degradation of e EF1A2 and activated c-Myc pathway by interacting with e EF1A2.However,whether SNX16 was involved in the replication of IAV remained unclear,and the role of ABTB1 in the interaction between SNX16 and e EF1A2 was also ill-defined.In this study,transient transfection of SNX16 in vitro significantly inhibited the replication of IAV.Moreover,plasmids expressing SNX16 with different tags obviously expressed two strips.To illustrate this phenomenon,we constructed mutation plasmids and found when the 144 position of SNX16 was mutated from R to A,only one strip was expressed,indicating that the R144 of SNX16 was the key amino acid for the expression of SNX16 with two strips.Confocal assay showed that R144 A mutation also affected the distribution characteristics of SNX16 in the cytoplasm of A549 cells from dot-like aggregation to dispersion.In addition,SNX16 R144 A mutation partially compensated for the proliferation titer of IAV by affecting the early stage of invasion of IAV.In summary,we revealed that ABTB1 targeted and degraded TRIM4 through proteasome system to preventing the degradation of NP protein caused by TRIM4 and finally positively regulated the nuclear import of vRNP of IAV.In addition,SNX16 significantly inhibited the replication of IAV by affecting the early stages of IAV invasion.SNX16 R144 A mutation affected the expression and distribution pattern of SNX16 and weaken its inhibition to IAV.Our research provided new ideas for the development of anti-IAV drugs.