| ObjectiveHBV infection is a complex process in which the virus and host factors interact with and influence each other.Host factors with antiviral activities are critical supplements to the host immune system.DExD/H-box proteins belong to the RNA helicases.Several DExD/H-box proteins have been reported to contribute to antiviral immunity.However,studies on the effect of DExD/H-box proteins on HBV replication are rare.We screened the DExD/H-box proteins to discover novel candidates with antiviral effect against HBV.Moreover,we elucidated the effects and molecular mechanism of DDX17 on the replication of HBV.Methods1.DExD/H-box genes were retrieved from NCBI/BLAST database.2.DExD/H-box genes amplified fom the cDNA derived from HepG2 cells with the use of RT-PCR were cloned into pCH9 vector to generate DExD/H-box expressing plasmids.3.HBV core promoter was cloned into a luciferase reporter vector to generate pCH9-pcore-Rluc.4.HepG2 cells were transiently cotransfected with DExD/H-box expressing plasmids and pCH9-pcore-Rluc.The luciferase activity was measured with Luciferase Assay System.5.HepG2 cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Western blot analysis.6.HepG2 cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Southern blot analysis.7.Huh7 cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Western blot analysis.8.Huh7 cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Southern blot analysis.9.HepG2 cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Northern blot analysis.10.Huh7 cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Northern blot analysis.11.ELISA analysis was used to test the levels of HBe Ag secreted from the HepG2 cells with DDX17 overexpressing.12.ELISA analysis was used to test the levels of HBe Ag secreted from the Huh7 cells with DDX17 overexpressing.13.ELISA analysis was used to test the levels of HBs Ag secreted from the HepG2 cells with DDX17 overexpressing.14.ELISA analysis was used to test the levels of HBs Ag secreted from the Huh7 cells with DDX17 overexpressing.15.RT-PCR analysis was performed to test the levels of the essential moleculars involved in ER-α,ZAP and IFN pathways.16.The A1004T+C1007G and A1077C+A1086C mutants of human DDX17 were generated.17.HepG2 cells were transiently cotransfected with DDX17 TPGRM,DDX17DEADM,wtDDX17 or vector and HBV1.3.Cells were lysed and subjected to Southern blot analysis or Northern blot analysis.18.HepG2 cell lysates overexpressing DDX17 were coincubated with the HBV ? stem loop and subjected to RNA-Protein Pull-Down analysis.19.The DDX17 gene knockout HepG2 cell lines were constructed by using CRISPR/Cas9.Gene deficiency was identified by sequencing and Western blot analysis.20.DDX17 KO cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Western blot analysis.21.DDX17 KO cells were transiently cotransfected with DDX17 and HBV1.3.Cells were lysed and subjected to Real-time PCR analysis and Southern blot analysis.22.DDX17 KO or Hep G2 cells were transiently cotransfected with pCH9-pcore-Rluc and CMV-Fluc.The luciferase activity was measured with the Dual-Luciferase Assay System.23.DDX17 KO cells were transiently cotransfected with DDX17,pCH9-pcore-Rluc and CMV-Fluc.The luciferase activity was measured with the Dual-Luciferase Assay System.24.The truncated mutants of HBV core promoter were generated.25.HepG2 cells were transiently cotransfected with pCH9-pcore-Rluc and CMV-Fluc.The luciferase activity was measured with the Dual-Luciferase Assay System.26.DDX17 KO or Hep G2 cells were transiently cotransfected with pCH9-BCP-Rluc and CMV-Fluc.The luciferase activity was measured with the Dual-Luciferase Assay System.27.DDX17 KO or Hep G2 cells were transiently cotransfected with pCH9-BCP-Rluc or pCH9-1635/1744-Rluc and CMV-Fluc.The luciferase activity was measured with Dual-Luciferase Assay System.Results1.Fifty-eight DExD/H-box genes were retrieved,including 16 DEAH/DExH and 42 DEAD/DExD subfamily members.2.Twenty-seven plasmids over-expressing DExD/H-box RNA helicases were constructed successfully.3.pCH9-pcore-Rluc was constructed successfully.4.Compared with the control,overexpression of DDX17 significantly inhibited HBV core promoter activity(P<0.0001).5.DDX17 plasmid could express DDX17 in HepG2 cells.6.Compared with the control,overexpression of DDX17 reduced HBV replication intermediates in HepG2 cells.7.DDX17 plasmid could express DDX17 in Huh7 cells.8.Compared with the control,overexpression of DDX17 reduced HBV replication intermediates in Huh7 cells.9.Compared with the control,stable expression of DDX17 reduced accumulation of HBV RNAs in HepG2 cells.10.Compared with the control,stable expression of DDX17 reduced accumulation of HBV RNAs in Huh7 cells.11.Compared with the control,overexpression of DDX17 significantly inhibited HBe Ag secreted into the supernatant of HepG2 cells(P=0.0032).12.Compared with the control,overexpression of DDX17 significantly inhibited HBe Ag secreted into the supernatant of Huh7 cells(P<0.0001).13.Compared with the control,overexpression of DDX17 significantly inhibited HBs Ag secreted into the supernatant of HepG2 cells(P=0.0004).14.Compared with the control,overexpression of DDX17 significantly inhibited HBs Ag secreted into the supernatant of Huh7 cells(P=0.0035).15.There was no difference in the mRNA levels of essential molecular involved in ER-α,ZAP or IFN pathways between the group overexpressing DDX17 or not.16.DDX17 mutants TPGRM and DEADM were constructed successfully.17.Overexpression of DDX17 TPGRM arised HBV replication intermediates and HBV RNAs,compared with wtDDX17.Whereas there was no difference in HBV replication intermediates and HBV RNAs between the DDX17 DEADM and wtDDX17 transfected groups.18.No interaction was detected between DDX17 and the HBV ? stem loop structure.19.Four DDX17 KO Hep G2 cell lines were established.20.Complementation with c-Myc-DDX17 in the DDX17 KO cells recoved the level of DDX17 expression.21.Compared with the control,overexpression of DDX17 reduced HBV replication intermediates in the DDX17 KO cells by Real-time PCR analysis(P=0.0101)and Southern blot analysis.22.Compared with the control,HBV core promoter activity in the DDX17 KO cells was significantly higher(P<0.0001).23.Compared with the control,overexpression of DDX17 in the DDX17 KO cells significantly inhibited HBV core promoter activity(P=0.0004).24.HBV core promoter truncations were constructed successfully.25.Compared with the control,expression of DDX17 in the DDX17 KO cells significantly inhibited HBV core promoter activity while activated core promoter mutants that lacking Enh Ⅰ and Enh Ⅱ or lacking Enh Ⅰ.However,Enh Ⅰ contributed 85%,while Enh Ⅱ contributed only 8.5%.Compared with the control,overexpression of DDX17 reduced Enh Ⅰ contribution by 11%,while overexpression of DDX17 arised Enh Ⅱ and BCP contribution slightly.26.There was no difference in the BCP promoter activity between the two cell lines.27.Compared with the control,1636 promoter activity in the DDX17 KO cells was significantly lower(P=0.0256)and 1635/1744 promoter activity in the DDX17 KO cells was significantly higher(P=0.0018).ConclusionWe identified that DDX17 gene can restrict HBV replication by a screen of DExD/H-box family proteins.DDX17 reduced HBV replication through suppressing the transcription of HBV RNAs by lowering the activity of Enh Ⅰ.The RNA bingding motif is important for the inhibiting effect of DDX17 on HBV replication. |
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