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Peptidylprolyl Cis/Trans Isomerases Parvulin 14 and Parvulin 17 Upregulates Hepatitis B Virus Replication

초록/요약

Part I Peptidylprolyl Cis/Trans Isomerases Parvulin 14 and Parvulin 17 Bind to HBx and cccDNA and Upregulate Hepatitis B Virus Replication from cccDNA to Virion in an HBx-Dependent Manner. The parvulin 14 (Par14) and parvulin 17 (Par17) proteins, which are both encoded by PIN4 gene, play roles in protein folding, chromatin remodeling, DNA binding, ribosome biogenesis, and cell cycle progression. However, the effects of Par14 and Par17 on viral replication have never been explored. In this study, we found that, in the presence of HBx, either Par14 or Par17 could upregulate hepatitis B virus (HBV) replication, whereas in the absence of HBx, neither Par14 nor Par17 had any effect on replication. Overexpression of Par14/Par17 markedly increased formation of covalently closed circular DNA (cccDNA), synthesis of HBV RNA and DNA, and virion secretion. Conversely, PIN4 knockdown significantly decreased HBV replication in HBV-transfected and -infected cells. Co-immunoprecipitation revealed that Par14/Par17 engaged in direct physical interactions with HBx in the cytoplasm, nucleus, and mitochondria, possibly mediated through substrate-binding residues on Par14/Par17 (E46/D74 and E71/D99, respectively) and conserved 19R20P-28R29P motifs on HBx. Furthermore, these interactions enhanced HBx stability, promoted HBx translocation to the nuclear and mitochondrial fractions, and increased HBV replication. Chromatin immunoprecipitation assays revealed that, in the presence of HBx,Par14/Par17 were efficiently recruited to cccDNA and promoted transcriptional activation via specific DNA-binding residues (S19/44). By contrast, in the absence of HBx, Par14/Par17 bound cccDNA only at the basal level and did not promote transcriptional activation. Taken together, our results demonstrate that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNA–Par14/17–HBx complex. Part II Parvulin 14 and Parvulin 17 Bind Both to Hepatitis B Virus Core Protein and Core Particle and Enhance HBV Replication. We reported recently that peptidylprolyl cis/trans isomerases parvulin 14 (Par14) and parvulin 17 (Par17) encoded by PIN4 gene upregulate HBV replication in an HBx-dependent manner by binding to conserved arginine–proline (RP) motifs on HBx. Since HBV core protein (HBc) has a conserved 133RP134 motif, we examined whether Par14/Par17 bind to HBc and/or core particle. Native agarose gel electrophoresis and immunoblotting and co-immunoprecipitation revealed that core particle binds to Par14/Par17 and core particle-defective, dimer-positive HBc-Y132A mutant can interact with Par14/Par17, respectively, demonstrating that Par14/Par17 interact with both core particle and HBc protein. Par14/Par17 interact with 133RP134 motif on HBc possibly via substrate-binding E46/D74 and E71/D99 motifs. Even though Par14/Par17 dissociated from core particle by heat-treatment, Par14/Par17 were still detected from opened-up core particle by 0.2 N NaOH treatment, demonstrating that Par14/Par17 bind onto and into the core particle. Furthermore, these interactions enhance the stabilities of HBc and core particle. Like HBc-Y132A, HBc-R133D or HBc-R133E mutants are also particle-defective and dimer-positive, demonstrating that the negatively charged residues at 133 cannot be tolerated for particle assembly. Even though the positively charged residue at 133 is solely important for Par14/17 interaction, 133RP134 motif is important for efficient HBV replication. Chromatin immunoprecipitation from HBV-infected cell revealed that HBc proteins are recruited onto cccDNA via E46/71 and D74/99 residues of Par14/Par17 and 133RP134 motif of HBc. Taken together, our results indicate that the interactions with HBc, Par14/Par17, and cccDNA in the nucleus and core particle–Par14/Par17 interactions in the cytoplasm are also important for HBV replication.

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목차

PART I 1
1. Introduction 2
2. Materials and Methods 6
2.1. Vector construction 6
2.2. Cell culture 10
2.3. DNA transfection 10
2.4. Establishment of stable cell lines 11
2.5. HBV virion analysis and in situ nucleic acid blotting 12
2.6. HBV preparation and infection 13
2.7. Core particle isolation and core particle immunoblotting 14
2.8. SDS-PAGE and immunoblotting 14
2.9. Northern and Southern blotting 15
2.10. Luciferase reporter assay 15
2.11. Nuclear, cytoplasmic, and mitochondrial fractionation 16
2.12. Juglone and PiB treatment 16
2.13. Co-immunoprecipitation 17
2.14. HBx stability analysis 17
2.15. cccDNA extraction 17
2.16. cccDNA chromatin immunoprecipitation and real-time PCR 18
2.17. HBx amino acid sequence alignment 20
2.18. Immunofluorescence analysis and confocal microscopy 20
2.19. Statistical analysis 21
3. Results 22
3.1. Parvulin inhibitors and PIN4 knockdown abrogate HBV replication 22
3.2. Overexpression of Par14 and Par17 augments HBV replication 27
3.3. Par14 and Par17 augment HBV cccDNA formation and promote viral transcription 31
3.4. Par14/Par17 upregulate HBV virion secretion 36
3.5. The S19, E46, and D74 residues of Par14 and S44, E71, and D99 residues of Par17 are important for Par14/Par17-mediated upregulation of HBV replication 40
3.6. Upregulation of HBV replication by Par14 or Par17 is HBx-dependent 47
3.7. Par14 and Par17 are novel binding partners of HBx 50
3.8. HBx RP motifs are crucial for Par14/Par17-mediated HBV replication 54
3.9. HBx RP motifs are Par14/Par17-interacting sites 60
3.10. E46 and D74 on Par14, and E71 and D99 on Par17, specifically interact with HBx 60
3.11. Interaction with Par14 and Par17 stabilizes HBx 65
3.12. Translocation of HBx to the nucleus or mitochondria is promoted by Par14 and Par17 69
3.13. Par14 and Par17 are recruited to HBV cccDNA, possibly through their S19/44 residues 73
4. Discussion 79
5. Conclusion 83
References 86
PART II 96
1. Introduction 97
2. Materials and Methods 101
2.1. Vector construction 101
2.2. Cell culture and DNA transfection 104
2.3. Stable cell line establishment 105
2.4. HBc amino acid sequence alignment 105
2.5. Core particle immunoblotting, SDS-PAGE and Western blotting 106
2.6. Co-immunoprecipitation 107
2.7. HBc stability analysis 108
2.8. Northern and Southern blotting 108
2.9. HBV preparation and infection 109
2.10. cccDNA extraction 110
2.11. cccDNA chromatin immunoprecipitation 110
2.12. Statistical analysis 112
3. Results 113
3.1. Par14 and Par17 bind to both HBV HBc protein and core particle 113
3.2. Par14/Par17 bind onto as well as inside of core particles 119
3.3. Substrate binding E46/71 and D74/99 residues of Par14/Par17 interact with HBc and/or core particle 120
3.4. HBc-R133 at the conserved RP motif is important for the interaction with Par14/Par17 125
3.5. Particle-defective HBc-R133D or -R133E mutants are defective for HBc–Par14/Par17 interaction 133
3.6. Interaction with Par14 and Par17 stabilizes HBc and core particle 138
3.7. HBc RP motif is crucial for Par14/Par17-mediated HBV replication 142
3.8. HBc–Par14/Par17–cccDNA interaction possibly via 133RP134 on HBc and E46/71 and D74/99 on Par14/Par17 148
4. Discussion 154
5. Conclusion 159
References 162

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