臺灣博碩士論文加值系統

B 型肝炎病毒(Hepatitis B Virus, HBV) 為一種會造成慢性肝炎、肝硬化及肝癌的血液性
病原體。HBV 進入細胞的機制目前並未完全了解，需被進一步的研究。最近藉由在
HepG2 細胞上表現sodium taurocholate cotransporting polypeptide (NTCP)的in vitro 感染
系統中，NTCP 已被證實為一個主要的receptor。然而這個感染系統需要依靠一個與生
理上不相關的化學物質polyethylene glycol (4% PEG) 。在HBV in vitro 感染的系統中，
高濃度的肝素(Heparin)被利用來做為一個抑制感染的抑制劑控制組。令人意外地，在
HepaRG 和HepG2 NTCP 的細胞中，生理濃度情況下的肝素可以藉由一個對Pre-S1
peptide 敏感，同時依靠NTCP 的方式促進HBV 感染。相較於肝素 的N-sulfation，
O-sulfation 對於促進HBV 的感染更為重要。這個建立於HepG2-NTCP-AS 細胞上的感
染系統能夠提供HBV genotype B 及C 以及從HBeAg 陽性及陰性的慢性帶原者血清中
獲得的病毒進行感染。綜合上述的結果，我們的研究提供了一個類似於自然界中HBV
感染人類的系統。此外，研究的結果也提供了一個在血液中肝素及與肝素結合的宿主因
子可能也參與HBV 病毒進入細胞的研究方向。

Hepatitis B virus (HBV) is a blood-borne pathogen responsible for chronic hepatitis, cirrhosis,
and liver cancer. The mechanism of HBV entry into hepatocytes remains to be investigated.
Recently, sodium taurocholate cotransporting polypeptide (NTCP) was discovered as a major
HBV receptor based on an in vitro infection system using NTCP-reconstituted HepG2 cells.
However, this infection system relies on the compound polyethylene glycol (4% PEG), which
is not physiologically relevant to human infection. High concentration of heparin has been
commonly used as an inhibitor control for in vitro infection in the field. Surprisingly, we found
that heparin at physiological concentration can enhance HBV infection in a PreS1-peptide
sensitive, NTCP-dependent manner in both HepaRG and HepG2-NTCP-AS cells. O-sulfation
of heparin is more important for the infection enhancement than N-sulfation. This system
based on the HepG2-NTCP-AS cells can support in vitro infection with HBV genotypes B and
C, as well as using serum samples from HBeAg positive and negative chronic carriers. In
summary, our study provides a PEG-free infection system closely resembling human natural
infection. In addition, it points to a future research direction for heparin and heparin-binding
host factor(s) in the blood, which are potentially involved in viral entry.

Acknowledgments - i
中文摘要 - ii
English Abstract -iii
Table of Contents - iv
List of Figures - vi
List of Tables - viii
Chapter 1 Introduction -1
1.1 Hepatitis B virus biology - 1
1.1.1 Classification of viruses within the Hepadnavirus family - 1
1.1.2 Virus structure - 2
1.1.3 Genome structure and organization - 2
1.1.4 The early events of HBV entry - 3
1.2 HBV in vitro infection systems - 4
1.3 Heparin effect on HBV entry - 5
1.4 Aim of the study - 6
Chapter 2 Results - 7
2.1 Establishment of an HBV in vitro infection system using a
HepG2-NTCP-AS cell line - 7
2.2 Heparin at physiological concentration in human plasma can stimulate
HBV in vitro infection - 8
2.3 Heparin facilitates PEG-free in vitro infection with HBV - 12
2.4 Effect of heparin sulfation for heparin-enhancement on HBV entry
- 13
Chapter 3 Discussion - 15
3.1 Heparin inhibition effect - 15
3.2 Heparin enhancement effect - 16
3.3 Possible mechanisms for heparin enhancement and inhibition - 18
Chapter 4 Methods - 20
4.1 Ethics statement - 20
4.2 Reagents - 20
4.3 Establishment of HepG2-NTCP-AS cell line - 21
4.4 Plasma fractionation - 21
4.5 HBV in vitro infection assay - 22
4.6 Western blot - 23
4.7 Immunofluorescence assay - 23
4.8 Southern blot assay - 24
4.9 HBsAg and HBeAg detection by ELISA - 24
References - 51

List of Figures

Fig 1. Establishment of an HBV in vitro infection system using a HepG2-NTCP-AS cell line. (Illustration of experimental design) - 26
Fig 2. Flag-tagged NTCP expression was detected by western blot in a HepG2-NTCP-AS cell line - 27
Fig 3. Increasing trend of both HBsAg and HBeAg confirms HepG2-NTCP-AS cells were infected with human HBV-containing serum - 28
Fig 4. HBV replicative intermediates were analysed by Southern blot assay - 29
Fig 5. HBc proteins were detected on HBV infected HepG2-NTCP-AS cells by IFA analysis - 30
Fig 6. HBV from both HBeAg positive and negative patients’ serum is infectious in vitro - 31
Fig 7. Heparin at physiological concentration in human plasma can stimulate HBV in vitro infection. (Illustration of experimental design) - 32
Fig 8. Human plasma, with or without heat pre-treatment, enhanced HBV in vitro infection - 33
Fig 9. Heparin is the principal ingredient in human plasma to the enhancement of HBV infection - 34
Fig 10. Heparin alone without human plasma can still enhance HBV infection - 35
Fig 11. Heparin at physiological concentrations (1 to 5 µg/ml) can enhance HBV infection in a dose-response experiment - 36
Fig 12. Treatment with PreS1 lipopolypeptide inhibited heparin-enhanced infection of HepG2-NTCP-AS cells - 37
Fig 13. Heparin-enhanced HBV infection can be abrogated by the continuous presence of a nucleoside analog 3TC (10uM), from 1 to 9 dpi - 38
Fig 14. Heparin facilitates PEG-free in vitro infection with HBV. (Illustration of experimental design) - 39
Fig 15. Heparin alone can enhance HBV infection in a PEG-independent manner - 40
Fig 16. A combinatory effect of heparin (4.5 µg/ml) and PEG (1.2%) can be detected by Southern blot analysis - 41
Fig 17. In a PEG-free in vitro infection system, low dose heparin (4.5 µg/ml) significantly increased the percentage of HBc-positive HepG2-NTCP-AS cells by confocal microscopy - 42
Fig 18. O-sulfation of heparin is required for its enhancement on HBV infection in HepG2-NTCP-AS cell system - 43
Fig 19. Heparin enhancement on HBV infection using the HepG2-NTCP-AS cells were validated by using a HepaRG infection system -44
Fig 20. Heparin can further enhance HBV infection using HepaRG cells in the presence of 1.2% or 4% PEG - 45
Fig 21. O-sulfation is also important for heparin enhancement of HBV infection in the HepaRG cell system - 46
Fig 22. Hypothetical mechanisms for the effects of heparin concentration on HBV entry via a two-step process - 47

List of Tables

Table 1. Clinical data of serum samples from Taiwanese HBV patients - 49
Table 2. Effects of heparin concentration on HBV in vitro infection - 50

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