| Influenza A virus is an important zoonotic pathogen,which has been the focus of veterinary medicine,virology and preventive medicine.IAV nuclear export protein(NEP),also known as NS2,is encoded by the fragment 8 of IAV by mRNA splicing.NEP is highly conserved in all subtypes of IAV strains,and its main function is to act as an adaptor mediating the nuclear export of vRNPs at the late stage of viral-replication cycle.It has also been reported that NEP can regulate the activity of polymerase and the synthesis of viral RNA.However,no more studies have been conducted to explain the mechanism of NEP regulating polymerase activity.Screening host proteins that interact with NEP,on the one hand,can reveal more about the biological function of NEP to virus replication.On the other hand,considering the conserved amino acid sequence of NEP,exploring new biological functions and explaining the mechanism can provide a theoretical basis for the research and development of anti-influenza drugs.Previous studies have presented that GPS2 is involved in regulating genome replication of papillomavirus and hepatitis C virus by interacting with viral proteins.In this study,GPS2 was shown to be a negative regulator of IAV replication,which interfered with the assembly of mature vRNPs by inhibiting the interaction between PB1 and PB2,and reduced the replication of viral genome RNA.In addition,IAV NEP interacted with GPS2 and mediated nuclear export of GPS2,thereby activating GPS2 protein degradation.Therefore,NEP-GPS2 interaction weakened the inhibition of GPS2 on viral polymerase activity and was beneficial to viral replication.The main content is as follows:1.NEP interacts with GPS2.Co-IP assay results showed that the IAV NEP protein interacted with host protein GPS2 under the overexpression condition,and this interaction was confirmed by GST-pull down assay.Co-IP also confirmed that the interaction between NEP and GPS2 in the IAV infected A549 cells.These results showed that NEP directly interacted with host protein GPS2,and this interaction was really existed in IAV infected cells.2.GPS2 inhibits the replication of IAV in cultured cells.In this study,in order to determine whether GPS2 affected IAV replication,specific siRNA targeting GPS2 was used to knock down GPS2 in A549 cells.Results showed that the viral titers of WSN/H1N1 and DW/H5N1 viruses were significantly enhanced in the GPS2-silenced cells,and the viral NP protein levels were significantly increased.While in GPS2-overexpressed A549 cells,the viral titers were decreased,suggesting that GPS2 was a negative regulator of IAV replication.Next,we constructed A549 cell lines with GPS2 knockout and demonstrated that GPS2 knockout significantly increased viral proliferation.In conclusion,GPS2 was a negative regulator to IAV proliferation.3.IAV infection induces degradation of GPS2 protein.In this study,we have determined whether IAV infection reduced the protein level of GPS2.Results showed no obvious change in the GPS2 mRNA level in WSN/H1N1-or DW/H5N1-infected A549 cells to mock infected A549 cells,while the protein levels of GPS2 decreased in a dose-dependent manner.The half-life assay results showed that viral infection significantly shortened the half-life of GPS2,and the degradation of GPS2 protein could be inhibited by MG132.Interestingly,NEP overexpression or virus infection could lead to nuclear export of GPS2 protein.Further studies showed that the degradation of GPS2 protein caused by virus infection or NEP overexpression was related to its nuclear output,and infected cells treated with LMB could inhibit the degradation.These results indicated that IAV infection promoted the degradation of GPS2 protein,which related to NEP-mediated nuclear export of GPS2.4.GPS2 reduces IAV polymerase activity by binding to vRNPs.In order to investigate the effect of GPS2 on IAV polymerase,a well-established mini-replicon assay system of WSN/H1N1 was applied.Results showed that GPS2 overexpression decreased polymerase activity in a dose-dependent manner,indicating that GPS2 negatively modulated IAV polymerase activity.Additionally,Co-IP assay results demonstrated that GPS2 could interact with proteins form RNPs(PB1,PB2 and NP)in IAV infected cells,and GPS2 directly reduced vRNP assembly by destroying the interaction between PB1 and PB2.Taken together,GPS2 interfered with the assembly of IAV polymerase,thereby inhibiting the activity of the polymerase.5.GPS2 participates in IAV genome replication by altering RNA synthesisTo further extend our observations,we investigated the effect of GPS2 on viral RNA synthesis.RT-qPCR analysis results showed that the viral RNA levels of all three species increased in the GPS2-silenced A549 cells compared with control cells at 12,24,and 36 hpi.Considering that IAV uses different strategies for transcription and for RNA replication,CHX was applied to define the effects of GPS2 on IAV genomicRNA transcription or replication.Results showed that GPS2 was involved in IAV genome replication.Taken together,these findings demonstrated that GPS2 reduced RNA synthesis in RNA replication but exerted no effect on primary transcription.6.NEP weakens the inhibition of GPS2 to viral polymerase activity.In this study,we tested the effects of NEP on the GPS2-inhibited polymerase activity of IAV.When co-transfected with 100 ng of NEP and 100 ng of GPS2,the inhibition effect to IAV polymerase activity caused by GPS2 was reduced in HEK293 T cells.These results provided evidence that the inhibition of GPS2 on IAV polymerase activity was independent of NEP.Moreover,the effects of LMB and MG132 on the inhibition of GPS2 to viral polymerase activity were studied because LMB and MG132 could block the degradation of GPS2 in IAV infected cells.MG132 treatment did not interfere with the rescue of luciferase activity by NEP between groups transfected with the same dose of GPS2,but LMB blocked it.Taken together,NEP weakened the inhibition of GPS2 to viral polymerase activity by interacting with GPS2 and mediating the nuclear export of GPS2. |
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