Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located...Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.展开更多
GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succini...GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succinimidyl-4-(N-maleimido-methyl) cyclo-hexane-1-carboxylate (SMCC) as crosslinking reagent to produce Fab'-HRP conjugate. A sandwich ELISA was established for the microquantitative determination of GST-π. The sensitivity was 11 pg/tube, which was far more sensitive than the radioimmunoassay so far reported. Using this method, the serum GST-π of 41 cases normal adult was found to be 1.06±0.94 ng/ml. The upper limit of the normal value was 2.6 ng/ml. In 30 cases of primary hepatocarcinoma, the level of serum GST-π was 24.4± 17.4 ng/ml, which was 23 times higher than the normal average value (P<0.01). The positive rate was 90%. In contrast, serum GST-π in 25 cases of chronic hepatitis was determined to be 1.74±1.16 ng/ml, which was not significantly different from the normal value (P>0.05). The pseudo-positive rate was 12.0%.展开更多
Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRFl-dependent GTPase. Polypeptide release...Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRFl-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation, In the present study, the C-terminal of Euplotes octocarinatus eRF3 （eRF3C） and truncate eRF3C lacking 76 amino acids in C-terminal （eRF3Ct） were expressed in Escherichia coll. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were purifled by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides interacted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRFla in Euplotes octocarinatus.展开更多
文摘Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.
文摘GST-π was purified from human placenta and its antiserum was raised in rabbits. The antibody IgC was purified and degraded into Fab' fragment which was conjugated with horseradish peroxidase (HRP) using N-succinimidyl-4-(N-maleimido-methyl) cyclo-hexane-1-carboxylate (SMCC) as crosslinking reagent to produce Fab'-HRP conjugate. A sandwich ELISA was established for the microquantitative determination of GST-π. The sensitivity was 11 pg/tube, which was far more sensitive than the radioimmunoassay so far reported. Using this method, the serum GST-π of 41 cases normal adult was found to be 1.06±0.94 ng/ml. The upper limit of the normal value was 2.6 ng/ml. In 30 cases of primary hepatocarcinoma, the level of serum GST-π was 24.4± 17.4 ng/ml, which was 23 times higher than the normal average value (P<0.01). The positive rate was 90%. In contrast, serum GST-π in 25 cases of chronic hepatitis was determined to be 1.74±1.16 ng/ml, which was not significantly different from the normal value (P>0.05). The pseudo-positive rate was 12.0%.
基金This work was supported by grants from National Natural Science Foundation of China(No.30670282,30470239,3030038)Natural Science Foundation of Shanxi Province(No.20041079,20051065).
文摘Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recognizes stop signals, whereas eRF3 is a ribosome-dependent and eRFl-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation, In the present study, the C-terminal of Euplotes octocarinatus eRF3 （eRF3C） and truncate eRF3C lacking 76 amino acids in C-terminal （eRF3Ct） were expressed in Escherichia coll. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were purifled by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides interacted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRFla in Euplotes octocarinatus.
