Pin1 Interacts with Hepatitis B Virus Core Particle and Enhances HBV Replication
- 주제(키워드) Hepatitis B virus , HBV replication , Core particle , PPIase Pin1 , Pin1-Core particle interaction
- 발행기관 아주대학교
- 지도교수 김경민
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033045
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Here, we demonstrate that the peptidyl-prolyl cis/trans isomerase Pin1 interacts noncovalently with the hepatitis B virus (HBV) core particle through phosphorylated serine/threonine-proline (pS/TP) motifs in the carboxyl-terminal domain (CTD) but not with particle-defective, dimer-positive mutants of HBc. This suggests that neither dimers nor monomers of HBc are Pin1-binding partners. The 162TP, 164SP, and 172SP motifs within the HBc CTD are important for the Pin1/core particle interaction. Although Pin1 dissociated from core particle upon heat treatment, it was detected as an opened-up core particle, demonstrating that Pin1 binds both to the outside and the inside of the core particle. Although the amino-terminal domain S/TP motifs of HBc are not involved in the interaction, 49SP contributes to core particle stability, and 128TP might be involved in core particle assembly, as shown by the decreased core particle level of S49A mutant through repeated freeze and thaw and low-level assembly of the T128A mutant, respectively. Overexpression of Pin1 increased core particle stability through their interactions, HBV DNA synthesis, and virion secretion without concomitant increases in HBV RNA levels, indicating that Pin1 may be involved in core particle assembly and maturation, thereby promoting the later stages of the HBV life cycle. By contrast, parvulin inhibitors and PIN1 knockdown reduced HBV replication. Since more Pin1 proteins bound to immature core particles than to mature core particles, the interaction appears to depend on the stage of virus replication. Taken together, the data suggest that physical association between Pin1 and phosphorylated core particles may induce structural alterations through isomerization by Pin1, induce dephosphorylation by unidentified host phosphatases, and promote completion of virus life cycle.
more목차
I. INTRODUCTION 1
II. MATERIALS AND METHODS 5
A. Plasmids Used in the Study 5
B. Cell culture and Transfection 7
C. Establishment of Stable Cell Lines 8
D. Immunoprecipitation, SDS-PAGE, and Immunoblotting 8
E. Native Agarose Gel Electrophoresis and Immunoblotting 9
F. Analysis of Extracellular HBV Particles 10
G. HBV Infection 11
H. Investigation of Pin1-Core Particle Interaction 11
I. In Vitro Analysis of Pin1-Core Particle Interaction and Phosphorylation Dependency 12
J. Stability Analysis of the Core Particle 12
K. Southern, Northern, and In Situ Nucleic Acid Blotting 12
L. Extraction of cccDNA 13
III. RESULTS 16
A. The HBV Core Particle Is a Novel Binding Substrate of Pin1 16
B. Dynamic On-off Interaction Between Pin1 and the HBV Core Particle 25
C. Pin1 Binds Both to the Outside and Inside of the HBV Core Particle 26
D. The HBc S/TP Motifs Are Highly Conserved Among Ten Genotypes of Human HBV 29
E. The HBc 162TP, 164SP, and 172SP Motifs Are Important for the Pin1/Core Particle Interaction 34
F. HBc NTD S/TP Motifs Contribute to Core Particle Stability and Assembly 38
G. The 16Ser and 34Trp Substrate Binding Residues of Pin1 Increase Core Particle Stability Through the Pin1/Core Particle Interaction 42
H. Inhibition of Parvulin or PIN1 KD Downregulates HBV Replication 47
I. Pin1 Overexpression Upregulates HBV Replication 52
J. Fewer Pin1 Proteins Bind to HBV WT Core Particles Than to RT-defective or Priming-deficient Core Particles 58
K. Pin1 Overexpression Increases Secretion of Virions, whereas PIN1 KD Decreases Secretion of Virions 61
IV. Discussion 68
References 71
