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Effects of Sirtuin 2 on Hepatitis B Virus Replication
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Kyongmin Kim | - |
| dc.contributor.author | PIRACHA ZAHRA ZAHID | - |
| dc.date.accessioned | 2019-04-01T16:42:41Z | - |
| dc.date.available | 2019-04-01T16:42:41Z | - |
| dc.date.issued | 2019-02 | - |
| dc.identifier.other | 28552 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/15247 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2019. 2 | - |
| dc.description.tableofcontents | Introduction 1 Materials and Methods 5 A. Vector construction 5 B. Cell culture, DNA transfection, and core particle immunoblotting 8 C. Stable cell lines 10 D. Northern, Southern, and nucleic acid blotting 11 E. SDS-PAGE and Western blotting 12 F. AGK2 treatment 13 G. Entecavir and lamivudine treatment 13 H. Reverse transcriptase (RT)-PCR 14 I. Co-immunoprecipitation 14 J. HBV preparation and infection 15 K. Fractionation of Sirt2 isoforms and HDAC6 16 L. Luciferase reporter assay 17 M. cccDNA extraction 17 N. cccDNA chromatin immunoprecipitation 18 O. Statistical analysis 20 Results 21 A. HBV replication upregulates endogenous Sirt2, leading to deacetylation of α-tubulin 21 B. Overexpression of Sirt2 isoform 1 upregulates HBV replication 29 C. Overexpression of Sirt2.1 increases HBV transcriptional activity 35 D. HBV replication is downregulated when Sirt2 is inhibited or knocked down 39 E. Overexpression of a Sirt2 dominant-negative mutant impairs HBV replication 46 F. Expression of replication-deficient mutant HBV impairs Sirt2 activity 46 G. Increased expression of Sirt2 in HBV-replicating cells activates the AKT/GSK-3β/β-catenin signaling pathway via the AKT/Sirt2 interaction 51 H. Sirt2-mediated upregulation of HBV replication is independent of HBx 55 I. AKT/GSK-3β/β-catenin signaling is downregulated in HBV replication-deficient mutant-transfected, entecavir-treated, Sirt2 DN mutant-overexpressing, and Sirt2-inhibited cells 60 J. Overexpression of Sirt2.5 downregulates HBV replication 67 K. Overexpression of Sirt2.5 decreases HBV transcriptional activity 71 L. Sirt2.5 was inactive for activating AKT/GSK-3β/β-catenin signaling pathway 77 M. Sirt2.5-mediated downregulation of HBV replication is independent of HBx 78 N. Nuclear export sequence and catalytic domains of Sirt2.1 are important for increasing HBV replication 83 O. Sirt2.1 and Sirt2.5 are recruited to HBV cccDNA 89 Discussion 92 References 98 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Effects of Sirtuin 2 on Hepatitis B Virus Replication | - |
| dc.title.alternative | PIRACHA ZAHRA ZAHID | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | PIRACHA ZAHRA ZAHID | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2019. 2 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 905152 | - |
| dc.identifier.uci | I804:41038-000000028552 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/common/orgView/000000028552 | - |
| dc.description.alternativeAbstract | Sirtuin 2 (Sirt2), a NAD+-dependent protein deacetylase, is overexpressed in many hepatocellular carcinomas (HCCs) and can deacetylate many proteins, including tubulins and AKT prior to AKT activation. Here, we found that endogenous Sirt2 was upregulated in hepatitis B virus (HBV) WT-replicating cells, leading to tubulin deacetylation; however, this was not the case in HBV replication-deficient mutant-transfected cells and 1.3mer HBV WT-transfected plus reverse transcriptase inhibitor (entecavir or lamivudine) treated cells but all HBV proteins are expressed. In HBV WT-replicating cells, upregulation of Sirt2 induced AKT activation, which consequently downregulated glycogen synthase kinase (GSK-3β) and increased β-catenin levels; however, downregulation of Sirt2 in HBV non-replicating cells impaired AKT/GSK-3β/β-catenin signaling. Overexpression of Sirt2 isoform 1 stimulated HBV transcription and consequently HBV DNA synthesis, which in turn activates AKT and consequently increases β-catenin levels, possibly through physical interactions with Sirt2 and AKT. Knockdown of Sirt2 by shRNAs or inhibition by AGK2 or dominant-negative mutant expression inhibited HBV replication, reduced AKT activation, and decreased β-catenin levels. The deacetylase inactive isoform 5 of Sirt2 (Sirt2.5) also showed inhibited HBV replication from transcription and failed to activate AKT/GSK-3β/β-catenin signaling. Both the Sirt2.1 and Sirt2.5 are recruited to cccDNA but Sirt2.5 showed more recruitment. Through HBV infection, we demonstrated that Sirt2 knockdown inhibited HBV replication from transcription. Although HBx itself activates AKT and upregulates β-catenin, Sirt2-mediated signaling and upregulated HBV replication were HBx-independent. Since constitutively active AKT inhibits HBV replication, the results suggest that upregulated Sirt2 and activated AKT may balance HBV replication to prolong viral replication, eventually leading to development of HCC. Also, the results indicate that Sirt2 inhibition may be a new therapeutic option for controlling HBV infection and preventing HCC. | - |
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