The Role Of The DDX Molecules And PC In The DENV-2 Replication

Dengue virus(DENV)is a positive-sense,single-stranded RNA virus,which is transmitted by Aedes mosquitoes.Each year 390 million people are infected with DENV,of which 96 million manifest clinical symptoms of infection.Among the four serotypes(DENV-1,DENV-2,DENV-3 and DENV-4),DENV-2 is the prevalent serotype in worldwide dengue epidemics.The DENV cause infection followed by a full spectrum of clinical manifestations,including asymptomatic infection,dengue fever,and dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS).At present,there is no specific antiviral drug available for the treatment of DENV infection.As a result of a evolution process for million years,viruses have developed sophisticated strategies to hijack and use cellular factors for entry,replication and propagation,alternate host transmission and to combat host cell defenses.When the virus proliferated in the host cell,some of the host molecules will induce the immune response to inhibit its replication.During the virus infection,the innate immune response is the first line of defense and the pattern recognition receptors(PRRs)are the important sensors.Retinoic acid-inducible gene I(RIG-I)and melanoma differentiation factor 5(MDA5),which belong to the RIG-I-like receptors(RLRs),are key PRRs in the DENV infection.And RLRs belong to the DEx D/H-box family of helicases,which are involved in the antiviral immune responses or the virus replication.In the previous study,some DExD/H-box helicases were over-expressed in the HEK 293 T cells infected with DENV-2,and the results showed that DDX21,DDX50 and DDX17 could inhibit or promote the virus replication.And the role of DDX21 in DENV-2 replication has been studied.DENV-2 NS3 protein is a multifunctional protein that plays an important role in the viral replication and the polyprotein precursor maturation,and this function is closely related to the interaction of the host proteins.Therefore,we first study the role of DDX50 and DDX17 in the DENV-2 replication.Then we use the affinity purification to screen the new host cell factors which could interact with the DENV-2 NS3 protein.And we study the role of the mitochondrial molecule pyruvate carboxylase(PC)in the DENV-2 replication.ObjectiveTo study the mechanism of DDX50,DDX17 and the new host molecules PC in the DENV-2 replication.Methods and Results 1.The role of DDX50 in the DENV-2 replicationTo determine whether DDX50 plays a role in DENV replication,we evaluated the impact of DDX50 down-regulation and over-expression on viral RNA replication.After DDX50 knock-down,HEK 293 T cells were infected with DENV-2(MOI=0.1 or MOI=1),which resulted in an increased DENV-2 RNA level.Over-expression of DDX50 was detected in HEK 293 T cells infected with DENV-2(MOI=0.1 or MOI=1),and the viral mRNA level decreased during early stages of DENV infection.Supernatants were collected from infected HEK 293 T cells(MOI=1)over-expressing DDX50,and viral titers were determined by plaque assay.The results showed that the viral titers were decreased in cells over-expressing DDX50.These results show that DDX50 could inhibit DENV-2 RNA replication during early stages of infection.To examine whether DDX50 regulates the production of IFN-? after DENV infection,we assayed IFN-? mRNA levels using qRT-PCR following DDX50 knock-down or over-expression in the cells.After DDX50 knock-down,the cells were infected with DENV-2,the level of IFN-? m RNA was found to have decreased during early stages of DENV-2 infection.In the HEK 293 T cells over-expressing DDX50,IFN-? mRNA levels were increased at 24 hrs post-DENV-2 infection.A luciferase reporter assay was performed to analyze the promoter activity of IFN-? in DDX50-overexpressing HEK 293 T cells following DENV-2 infection.Our results revealed that over-expression of DDX50 enhanced the level of transcription from the IFN-? promoter.Over-expression of DDX50 in the cells infected with DENV-2 increased transcription from the IFN-? promoter.IFN-? luciferase activity was also increased after poly(I:C)stimulation.After DDX50 was co-expressed with the NF-?B reporter plasmid pNF-?B-luc in HEK 293 T cells,transcription from the NF-?B promoter was significantly increased.These data suggest that DDX50 inhibited viral RNA synthesis by up-regulating IFN-? production.To examine whether DDX50 is involved in these pathways to enhance IFN-? production,we co-transfected HEK 293 T cells with pEGFP-DDX50 and pFlag-RIG-I or pEF-MDA5 and IFN-? luciferase reporter plasmids.At 48 hrs post-transfection,transcription from the IFN-? promoter was increased in HEK 293 T cells,which were co-transfected with plasmids encoding DDX50 and either RIG-I or MDA5.When the co-transfected HEK 293 T cells were infected with DENV-2,the level of IFN-? transcription was also increased.These data indicated that DDX50 exerts an additive effect on IFN-? promoter activation with RIG-1 or MDA5 in DENV-infected cells.To examine whether DENV-2 virus infection triggers the translocation of DDX50,we adopted an immunofluorescence assay.DENV-2 infection did not result in translocation of DDX50 from the nucleus to cytoplasm.Therefore,we speculate that DDX50 may act as a co-transcription factor for IFN-? in the nucleus.Lastly,we examined which types of ISGs were involved in the alteration of the DENV-2 virus-infected cells that over-expressed DDX50.First,we analyzed the transcriptional level of interferon-stimulated response element(ISRE)in DDX50-overexpressing HEK 293 T cells infected with DENV-2.We found that DDX50 over-expression increased the transcriptional level of ISRE promoter-driven luciferase reporter after DENV infection.HEK 293 T cells were then infected by DDX50 lentivirus following DENV-2 infection,we found that the levels of Mx1,OAS1,IFITM3 and ISG15 mRNA were increased.Thus,DDX50 inhibited DENV-2 infection by enhancing the production of several ISGs.2.The role of DDX17 in the DENV-2 replicationTo determine whether DDX17 plays a role in DENV replication,we evaluated the impact of DDX17 down-regulation and over-expression on viral RNA replication.After DDX17 knock-down,HEK 293 T cells were infected with DENV-2,which resulted in a decreased DENV-2 RNA level.Over-expression of DDX17 was detected in HEK 293 T cells infected with DENV-2,and the viral mRNA level increased during early stages of DENV infection.These results show that DDX17 could promote DENV-2 RNA replication during early stages of infection.To examine whether DDX17 regulates the production of IFN-? after DENV-2 infection,a luciferase reporter assay was performed to analyze the promoter activity of IFN-? in DDX17-overexpressing HEK 293 T and He La cells.Our results revealed that over-expression of DDX17 inhibited the level of transcription from the IFN-? promoter.Over-expression of DDX17 in the cells infected with DENV-2 decreased transcription from the IFN-? promoter.These data suggest that DDX17 may promote viral RNA synthesis by inhibiting IFN-? production.To clarify whether DDX17 interacts with DENV-2 proteins or viral RNA,we adopted an immunofluorescence assay and a RIP assay.The results showed that DDX17 was distributed in the nucleus and cytoplasm in HEK 293 T cells infected with DENV-2,and it was colocalized with the DENV-2 dsRNA.We also observed that DDX17 was colocalized with DENV-2 NS3 in the cytoplasm.Thus we speculate that DDX17 may be located in the viral replication complex.The results of RIP assay showed that DENV-2 RNA fragment could be amplified in the experimental group compared with the control group.This result indicates that DDX17 could bind DENV-2 RNA,so we hypothesize that DDX17 may be involved in the viral replication.In DENV-2 infected HEK 293 T cells,we found that the G3BP1 protein is dispersed in the cytoplasm,and we did not find the SG formation.But in the infected cells,we found that DENV-2 dsRNA,DDX17 and G3BP1 were colocalized in the cytoplasm,and DENV-2 NS3,DDX17 and G3BP1 were colocalized in the cytoplasm.The results suggest that DDX17 and G3BP1 are likely to be involved in viral replication and are located in the viral replication complex.The results of immunoprecipitation showed that DDX17 could interact with G3BP1.Over-expression of DDX17 and G3BP1 in HEK 293 T cells infected with DENV-2(MOI=1),and the viral mRNA level increased during early stages of DENV infection.The plaque assay results showed that the viral titers were increased in cells over-expressing DDX17 and G3BP1.These results show that over-expressing DDX17 and G3BP1 could promote DENV-2 RNA replication during early stages of infection.3.The role of PC in the DENV-2 replicationHEK 293 T cells were transfected with the plasmid pCI-NS3-SF-TAP,and the affinity purifications were done using standard conditions according to the TAP strategy.The eluted samples were applied on SDS-PAGE gel and specific bands were identified by LC-MS/MS.Through the data analysis,PC,HSPD1,FARSA and Vimentin could interact with DENV-2 NS3 protein.And PC was selected as the research object to further study.To clarify whether PC interacts with DENV-2 NS3,we adopted an immunofluorescence assay and an immunoprecipitation assay.The results of laser confocal microscopy showed that PC was colocalized with NS3 in DENV-2 infected A549 and HEK 293 T cells.The results of immunoprecipitation showed that PC interacted with DENV-2 NS3,and NS3 could interact with the CT region of PC.To determine whether the PC plays a role in DENV replication,we evaluated the impact of PC over-expression on viral RNA replication.Over-expression of PC was detected in A549 cells infected with DENV-2(MOI=0.5/1),and the viral mRNA level decreased at 12 hrs.Over-expression of PC was detected in HEK 293 T cells infected with DENV-2(MOI=0.05/0.1/0.5/1),and the viral mRNA level increased at 12 and 24 hrs.These results show that PC could inhibit DENV-2 RNA replication in A549 cells and promote DENV-2 RNA replication in HEK 293 T cells during early stages of infection.These results suggest that PC may have different effects on DENV-2 replication in different cell lines.To examine whether PC regulates the production of IFN-? after DENV-2 infection,luciferase reporter assays were performed to analyze the promoter activity of IFN-? and NF-?B in PC-overexpressing A549 and HEK 293 T cells.Our results revealed that over-expression of PC promoted the level of transcription from the IFN-? promoter and NF-?B promoter in A549 cells infected with DENV-2.But in HEK 293 T cells,over-expression of PC in the cells infected with DENV-2 did not promoter the transcription from the IFN-? promoter and NF-?B promoter.Therefore,PC may have different effects on IFN-? induction in different cell lines.The results of laser confocal microscopy showed that PC was colocalized with NS3 on the mitochondria in DENV-2 infected A549 and HEK 293 T cells.The results suggest that DENV-2 may affect the immune response through the interaction of NS3 and PC on mitochondria.A549,HEK 293 T and Huh7 cells were infected with DENV-2 for 48 h,and the mitochondria were stained with the MitoTracker probe.The results of laser confocal microscopy showed that the mitochondria were shortened in DENV-2 infected A549 cells,but the mitochondria were elongated in DENV-2 infected HEK 293 T cells and Huh7 cells.These results suggest that the effect of DENV-2 infection on mitochondrial morphology is different in different cells lines.Conclusion 1.DDX50 inhibits DENV-2 replication during early stages of infection by up-regulating IFN-? production.Furthermore,DDX50 activates the IFN-? promoter in conjunction with RIG-I or MDA5 in DENV-2-infected cells.Finally,over-expression of DDX50 in virus-infected cells enhances the production of several ISGs.2.DDX17 promotes DENV-2 replication during early stages of infection by inhibiting IFN-? production.Furthermore,DDX17 could interact with the DENV-2 dsRNA and participate in the virus replication.DDX17 could interact with G3BP1,and over-expressing DDX17 and G3BP1 could promote DENV-2 RNA replication during early stages of infection.3.DENV-2 NS3 could interact with PC during early stages of infection,and the binding region is the CT functional domain of PC.And PC may have different effects on DENV-2 replication in different cell lines.In A549 cells,PC inhibits DENV-2 replication during early stages of infection by up-regulating IFN-? production,and the interaction of NS3 and PC on mitochondria may induce mitochondrial autophagy to inhibit DENV-2 replication.In HEK 293 T cells,the interaction between NS3 and PC on mitochondria may affect the activity of PC or other proteins,thus affecting the function of mitochondria and promoting the DENV-2 replication.