| The viral ribonucleoprotein(vRNP)of the influenza A virus(IAV)is responsible for the viral RNA transcription and replication in the nucleus.It is a very important process of vRNP entering the nucleus from the cytoplasm in the IAV life cycle,and the process need many host proteins,and there are few studies of host proteins in this aspect.Studying the host proteins involved in vRNP nuclear entry can not only reveal the molecular mechanisms of the nuclear import of IAV vRNP,but also provide potential targets for antiviral drug development.Previous study has indicated that eukaryotic translation elongation factor 1 ?(eEF1D)may associate with RNP subunits,but its roles in IAV replication are unclear.Herein,we showed that eEF1D was an inhibitor of IAV life cycle,because knockout of eEF1D resulted in a significant increase in virus yield.eEF1D interacted with all RNP subunits and impeded the nuclear import of NP and PA-PB1 heterodimer of IAV,resulting in inhibiting the vRNP assembly,polymerase activity and viral RNAs synthesis of IAV.The main research contents are as follows: 1.eEF1D inhibits IAV replicationThis study examined the effect on IAV replication by overexpressing eEF1D.Experimental results exhibited that eEF1D inhibited the replication of IAV.Next,we used si RNA and CRISPR/ Cas9 system to knock down and knock out endogenous eEF1D in A549 cells,and detected the impact on human influenza virus PR8/ H1N1,avian influenza virus HM/ H5N1,and swine influenza virus HB/ H1N1 replication,respectively.Experimental results showed that knockdown or knockout of eEF1D could significantly up-regulate the titers of PR8 / H1N1,HM / H5N1 and HB / H1N1 viruses,and also could increase the synthesis of NP protein,indicating that eEF1D is a negative regulator of IAV.2.eEF1D interacts with vRNPResults of co-immunoprecipitation(Co-IP)experiments showed that eEF1D interacted with all subunits of RNP(PA,PB1,PB2,and NP)under transfection,and also with NP in an RNA-dependent manner.Further studies of infected cells with IAV confirmed the real interactions between viral PA,PB1,PB2,NP and eEF1D,and the interaction between NP and eEF1D was mediated by RNA.In addition,eEF1D co-localized with PA,PB1,and NP in the cytoplasm and the cytoplasmic retention of NP was appeared in transfected cells.Also,eEF1D co-localized with PA,PB1,NP,and a small amount of PB2 in the cytoplasm in infected cells.These results show that eEF1D disturbs the distribution of NP,implying a vital role in regulating the nuclear import of vRNP subunits.3.eEF1D impedes the nuclear import of NP and PA-PB1 heterodimerThe effect of eEF1D on the nuclear import of IAV NP was detected.The data of indirect immunofluorescence assay and nuclear and cytoplasmic fractionation assay demonstrated that eEF1D blocked the nuclear import of IAV NP.And the inhibition of eEF1D was appeared by destroying the interaction between NP protein and importin ?5.Because of different ways of the nuclear import of RNP subunits,we also detected the effect of eEF1D on the nuclear import of viral PA-PB1 heterodimer and PB2.Experimental results showed that eEF1D inhibited the nuclear import of PA-PB1 heterodimer of IAV by interfering with the interaction between PB1 and Ran BP5,but had no effect on the nuclear import of PB2.These results suggest that eEF1D can hinder the nuclear entry of new vRNP of IAV.4.eEF1D inhibits the nuclear import of vRNPThe effect of eEF1D on the nuclear import of IAV vRNP was examined.The endogenous eEF1D was knocked down or knocked out by specific si RNA or CRISPR/Cas9 system.And the localization of NP was detected with or without CHX treatment in PR8/H1N1 infected cells of eEF1D knockdown or knockout cells at 2 hpi.Results of confocal experiments and statistical analysis have proven that knockdown or knockout of eEF1D could significantly promote the nuclear import of vRNP,whether CHX treated or not.The same results were also obtained in the protein level when eEF1D knocked out,suggesting that eEF1D could inhibit the vRNP entry process of IAV.5.eEF1D suppresses vRNP assembly and RNAs synthesisFurther study shows that eEF1D inhibits the IAV oligomerization of NP and disrupts the formation of polymerase PA,PB1,and PB2 ternary complexes,which ultimately affects the vRNP assembly.vRNP is the smallest functional unit of IAV transcription and replication.Then,we examined the effects of eEF1D overexpression and knockout on v RNA,c RNA,and m RNA of IAV.Results of real-time PCR showed that overexpression of eEF1D significantly reduced the synthesis of v RNA,c RNA,and m RNA of IAV,while knockout of eEF1D significantly increased the synthesis of IAV RNAs,indicating that the transcription and replication of IAV were affected.6.Both N-and C-terminus of eEF1D can regulate IAV replicationAs a translation elongation factor,eEF1D has two important functional domains: the leucine zipper region at the N-terminus,which mainly plays a role in protein binding;the guanine nucleotide exchange factor region at the C-terminus,which mainly catalyzes the exchange of GDP and GTP.In order to identify the important regions of eEF1D that regulate IAV replication,we divided eEF1D into eEF1D-1(N-terminus)and eEF1D-2(C-terminus).The results of indirect immunofluorescence experiments showed that the subcellular localization of eEF1D-1(N-terminus)and eEF1D-2(C-terminus)just like full length did not change,all in the cytoplasm.And the results of dual luciferase assay demonstrated that both eEF1D-1 and eEF1D-2 inhibited the polymerase activity of IAV.Real-time PCR experiments further revealed that eEF1D-1 and eEF1D-2 could significantly decrease the m RNA level of IAV NP.These experimental data indicate that both N-and C-terminus of eEF1D play roles in the regulation of IAV replication.Together,this study demonstrates that eEF1D can interact with vRNP and negatively regulate the IAV replication by inhibition of the nuclear import of RNP subunits,which not only uncovers a novel role of eEF1D in IAV replication,but also provides new insights into the mechanisms of nuclear import of vRNP proteins. |
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