AIM: To characterize the nuclear import of hepatitis B virus (HBV) polymerase (P) and its relevance for the viral life cycle.METHODS: Sequence analysis was performed to predict functional motives within P. Phosphoryla...AIM: To characterize the nuclear import of hepatitis B virus (HBV) polymerase (P) and its relevance for the viral life cycle.METHODS: Sequence analysis was performed to predict functional motives within P. Phosphorylation of P was analyzed by in vitro phosphorylation. Phosphorylation site and nuclear localization signal (NLS) were destroyed by site directed mutagenesis. Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding. Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV.RESULTS: We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinase II (CKII) phosphorylation site located within the terminal protein domain (TP) of the HBV polymerase. Inhibition of CKII impairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase. Binding of the import factor karyopherin-α2 to the polymerase depends on its CKII-mediated phosphorylation of the bipartite NLS. In HBV-infected primary Tupaia hepatocytes CKII inhibition in the early phase (post entry phase) of the infection process prevents the establishment of the infection.CONCLUSION: Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.展开更多
Objective:Tea polyphenols are natural extracts used widely throughout the world.However,the severe astringency of tea polyphenols has reduced patient compliance.Based on the analysis of the formation mechanism of astr...Objective:Tea polyphenols are natural extracts used widely throughout the world.However,the severe astringency of tea polyphenols has reduced patient compliance.Based on the analysis of the formation mechanism of astringency,this paper hopes to propose a new method to control the astringency of tea polyphenols and improve patient compliance without changing its effect.Methods:Artificial saliva was used to prepare the tea polyphenols solution with different p H,using bcasein to imitate salivary protein,and preparing 1.2 mg/m L b-casein solution.A fluorescence quenching test was used to study the interaction between tea polyphenols and b-casein,combined with the stability test results of the compound,we can choose the p H with weak binding but good stability as the best p H for masking astringency.The taste-masking tablets were prepared under the best p H conditions,and the Xinnaojian Original Tablets were prepared according to the conventional preparation method.The disintegration time limit and solubility were tested respectively.The astringency of Xinnaojian original tablets and taste-masking tablets was evaluated by visual analogue scale(VAS).Results:The result of the fluorescence quenching test prompted that the combination force was the weakest when the p H was 4.9.Further synchronous fluorescence analysis showed that an increase in p H resulted in a decrease of the binding sites between tea polyphenols and b-casein,and this decrease was closely related to changes in tryptophan residues in b-casein.Both original and taste-masking Xinnaojian Tablets were prepared.Volunteers’VAS scores illustrated that the astringency improved significantly with the masking tablets(P<0.05).Conclusion:This p H-adjusting masking treatment had little effect on the recovery of polyphenols from the tablets or the dissolution of the tablets.This study provides a novel and feasible astringency masking technology for tea polyphenols and its preparation.展开更多
文摘AIM: To characterize the nuclear import of hepatitis B virus (HBV) polymerase (P) and its relevance for the viral life cycle.METHODS: Sequence analysis was performed to predict functional motives within P. Phosphorylation of P was analyzed by in vitro phosphorylation. Phosphorylation site and nuclear localization signal (NLS) were destroyed by site directed mutagenesis. Functionality of the identified NLS was analyzed by confocal fluorescence microscopy and characterizing the karyopherin binding. Relevance of the structural motives for viral life cycle was studied by infection of primary Tupaia hepatocytes with HBV.RESULTS: We identified by sequence alignment and functional experiments a conserved bipartite NLS containing a casein kinase II (CKII) phosphorylation site located within the terminal protein domain (TP) of the HBV polymerase. Inhibition of CKII impairs the functionality of this NLS and thereby prevents the nuclear import of the polymerase. Binding of the import factor karyopherin-α2 to the polymerase depends on its CKII-mediated phosphorylation of the bipartite NLS. In HBV-infected primary Tupaia hepatocytes CKII inhibition in the early phase (post entry phase) of the infection process prevents the establishment of the infection.CONCLUSION: Based on these data it is suggested that during HBV infection the final import of the genome complex into the nucleus is mediated by a novel bipartite NLS localized in the TP domain of HBV polymerase.
基金supported by the National Natural Science Foundation of China.(Nos.81673615,81403115)。
文摘Objective:Tea polyphenols are natural extracts used widely throughout the world.However,the severe astringency of tea polyphenols has reduced patient compliance.Based on the analysis of the formation mechanism of astringency,this paper hopes to propose a new method to control the astringency of tea polyphenols and improve patient compliance without changing its effect.Methods:Artificial saliva was used to prepare the tea polyphenols solution with different p H,using bcasein to imitate salivary protein,and preparing 1.2 mg/m L b-casein solution.A fluorescence quenching test was used to study the interaction between tea polyphenols and b-casein,combined with the stability test results of the compound,we can choose the p H with weak binding but good stability as the best p H for masking astringency.The taste-masking tablets were prepared under the best p H conditions,and the Xinnaojian Original Tablets were prepared according to the conventional preparation method.The disintegration time limit and solubility were tested respectively.The astringency of Xinnaojian original tablets and taste-masking tablets was evaluated by visual analogue scale(VAS).Results:The result of the fluorescence quenching test prompted that the combination force was the weakest when the p H was 4.9.Further synchronous fluorescence analysis showed that an increase in p H resulted in a decrease of the binding sites between tea polyphenols and b-casein,and this decrease was closely related to changes in tryptophan residues in b-casein.Both original and taste-masking Xinnaojian Tablets were prepared.Volunteers’VAS scores illustrated that the astringency improved significantly with the masking tablets(P<0.05).Conclusion:This p H-adjusting masking treatment had little effect on the recovery of polyphenols from the tablets or the dissolution of the tablets.This study provides a novel and feasible astringency masking technology for tea polyphenols and its preparation.
