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Peptidylprolyl Cis/Trans Isomerases Parvulin 14 and Parvulin 17 Upregulates Hepatitis B Virus Replication
- Author(s)
- SAEED UMAR
- Advisor
- Kyongmin Kim
- Department
- 일반대학원 의생명과학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2020-08
- Language
- eng
- Alternative Abstract
- 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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- Graduate School of Ajou University > Department of Biomedical Sciences > 4. Theses(Ph.D)
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