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2011 추계 STS 김진욱 Basic in Hepatitis B Virus-Cccdna Activity in HBV Infection B..Pdf Covalently closed circular DNA activity in HBV infection 분당서울대학교병원 내과 김 진 욱 Covalently closed circular DNA (cccDNA) is a viral minichromosome from which HBV RNA replication intermediate (pregenomic RNA) is transcribed. Intrahepatic contents of cccDNA is lower in HBeAg-negative CHB compared to HBeAg-positive CHB, but cccDNA persists even after HBsAg loss (occult HBV infection). There are evidences indicating that activity of cccDNA differ in different stages of CHB infection. Possible genetic and epigenetic mechanisms responsible for the modulation of cccDNA activity are discussed in this review. Key words: Covalently closed circular DNA, Chronic hepatitis B, Hepatitis B virus 서 론 HBV covalently closed circular DNA (cccDNA)는 간세포 핵 내에 존재하는 double-stranded circular DNA 로서, HBV 증식에 필수적인 RNA intermediate인 pregenomic RNA (pgRNA)의 template로 작용하며 항바 이러스 약제 중단 시 HBV가 다시 증식하게 하는 주 원인이다.1 HBV cccDNA의 정성 및 정량 검사법 HBV cccDNA를 정제하기 위하여 우선 chromosomally integrated linear HBV DNA를 제거할 필요가 있다. 이를 위하여 SDS/NaCl을 이용한 differential precipitation (Hirt method)을 통하여 protein-rich cellular DNA를 침전시키고 상층액에서 cccDNA fraction을 얻는다.2 여기에 Plasmid-safe DNase®를 처리하면 circular double stranded DNA만 남기고 cellular DNA는 선택적으로 제거되어 genomic DNA의 contamination을 재차 차단 한다. 하지만 Plasmid-safe DNase®로 relaxed circular DNA (rcDNA)를 완전히 제거할 수는 없으며, rcDNA 에 존재하는 gap을 중간에 포함하도록 primer를 제작, 이용하는 cccDNA-specific PCR로 cccDNA를 검출, 정량한다.3,4 8 김진욱 ❚ Covalently closed circular DNA activity in HBV infection CHB의 자연경과 중 간세포 내 HBV cccDNA copy number의 변화 간 조직검사 검체에서 HBV cccDNA를 real-time PCR로 정량한 연구에서 cccDNA는 HBeAg+ CHB에서 HBeAg- CHB에 비하여 더 높으며3,5 HBeAg- CHB와 inactive carrier간에는 차이를 보이지 않았다.3 흥미롭 게도 HBsAg이 소실된 환자에서도 미량이지만 cccDNA는 검출되었다.3 혈청에서 HBsAg이 검출되지 않 지만 간조직에 HBV DNA가 존재하는 occult hepatitis B virus infection (OBI)은 최근에 그 개념이 정립되 었으며6 핵 내 잔존하는 cccDNA가 그 원인으로 생각된다.7 항 바이러스 치료 후 간세포 내 HBV cccDNA copy number의 변화 Adefovir를 48주 간 치료 후 간 세포에서 cccDNA는 유의하게 감소함이 보고되었다(-0.80 log10 copies/cell, 84% reduction)3. Lamivudine과 entecavir를 1년간 투여한 환자를 대상으로 한 연구에서도 후 간조직 내 cccDNA는 감소 소견을 보였다.4,8 그러나 이러한 연구는 소수의 환자를 대상으로 하였으며, cccDNA가 유의하게 감소하지 않았다는 보고도 있어9 더 많은 수의 환자를 대상으로 추가 연구가 필요하다. 한편, 치료 종료 시점에서 간조직 내 cccDNA의 양이 sustained virologic response를 예측할 수 있다는 연구가 있다.10 CHB의 자연경과 중 HBV cccDNA activity의 변화 및 그 조절기전 간세포 내 HBV DNA양의 결정요인으로서 HBV cccDNA copy number 이외에도 cccDNA의 transcriptional activity가 영향을 줄 가능성이 있다. 그 근거로서, 1) HBeAg+ CHB의 경우 cccDNA와 혈청 HBV DNA 사이 양의 상관관계를 보이는 반면 HBeAg- CHB의 경우 상관관계를 보이지 않으며 cccDNA activity를 반영하 는 virion productivity index (cytoplasmic rcDNA/cccDNA)가 감소되어 있는점,11 2) 상술하였듯이 OBI에서 간세포 내에 HBV cccDNA가 존재하여, 어떤 방식으로든 cccDNA의 transcriptional activity가 억제되고 있 음을 시사함12 등이다. 아직까지 cccDNA의 transcriptional activity가 조절되는 기전은 확립되지 않았으나 host immune mediated factor와 viral factor가 기여할 것으로 생각되며, viral factor는 다시 genetic change와 epigenetic factor로 구분하여 생각해볼 수 있다.7 1. Host immune factor HBV pgRNA의 transcription은 basal core promoter (BCP)와 core upstream regulatory sequence (CURS) 및 enhancer II의 조절을 받으며, 많은 transcription factor가 이들 부위에 결합하여 조절기능을 나타낸다.13 9 2011년 대한간학회 추계 Single Topic Symposium Host immunity가 여러 transcriptional factor를 조절함으로써 cccDNA transcriptional activity에 영향을 줄 가 능성이 충분히 예상되며 intracellular cytokine애 의한 non-cytolytic mechanism이 cccDNA activity를 조절함이 acute HBV infection model에서 시사되었으나14 CHB에서 noncytolytic control mechanism에 대해서는 아직 별로 알려진 바가 없다. 2. Genetic change 실험적으로 HBV sequence에 mutation을 유도하면 HBV replication이 억제됨15으로 미루어 자연적으로 유발된 돌연변이가 HBV cccDNA activity를 조절할 가능성을 생각해볼 수 있으나, 이후 in vitro study에 서 precore mutation (G1896A, G1899A)은 viral replication에 유의한 영향을 보이지 않았다.16,17 BCP의 double mutation (A1762T/G1764A)이 HBV 증식에 미치는 영향에 대한 연구는 pgRNA의 transcription이 증 가했다는 보고와18,19 유의한 영향이 없다는 상충된 결과를 보였다.17 3. Epigenetic control DNA와 결합하는 histone의 modification (i.e. acetylation)과 DNA methylation은 유전자의 발현을 조절하 는 가장 중요한 epigenetic mechanism이다. HBV cccDNA activity의 epigenetic control은 비교적 최근에 알 려지기 시작했으며, cccDNA-bound histone의 acetylation여부가 cccDNA의 activity에 영향을 미침이 알려 졌으며20 HBx 단백이 histone acetylation의 조절인자이다.21 한편, cellular DNA에 삽입된 HBV DNA는 메 틸화가 일어남이 보고되었으며,22 이 메틸화된 HBV DNA는 발현이 억제됨이 알려진 바 있는데,23-26 최근 HBV cccDNA에서도 유사한 메틸화가 일어남이 보고되었으며27,28 메틸화된 HBV cccDNA는 transcriptional activity가 저하됨이 알려졌다.29 결 론 HBV cccDNA의 activity가 조절되는 기전은 아직 충분히 밝혀지지 않았으며, 이에 대한 연구는 HBV replication에 대한 이해를 증진시킬 뿐 아니라 HBV cccDNA activity의 조절을 통한 새로운 기전의 항바 이러스 치료법을 개발하는 중요한 시발점이 될 수 있을 것이다. 참고문헌 1. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev 2000;64:51-68. 2. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 1967;26:365-369. 3. Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, et al. Persistence of cccDNA 10 김진욱 ❚ Covalently closed circular DNA activity in HBV infection during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 2004;126:1750-1758. 4. Bourne EJ, Dienstag JL, Lopez VA, Sander TJ, Longlet JM, Hall JG, et al. Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy. J Viral Hepat 2007;14:55-63. 5. Wong DK, Yuen MF, Yuan H, Sum SS, Hui CK, Hall J, et al. Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. Hepatology 2004;40:727-737. 6. Raimondo G, Allain JP, Brunetto MR, Buendia MA, Chen DS, Colombo M, et al. Statements from the Taormina expert meeting on occult hepatitis B virus infection. J Hepatol 2008;49:652-657. 7. Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. Control of cccDNA function in hepatitis B virus infection. J Hepatol 2009;51:581-592. 8. Wong DK, Yuen MF, Ngai VW, Fung J, Lai CL. One-year entecavir or lamivudine therapy results in reduction of hepatitis B virus intrahepatic covalently closed circular DNA levels. Antivir Ther 2006;11:909-916. 9. Cheng PN, Liu WC, Tsai HW, Wu IC, Chang TT, Young KC. Association of intrahepatic cccDNA reduction with the improvement of liver histology in chronic hepatitis B patients receiving oral antiviral agents. J Med Virol 2011;83:602-607. 10. Sung JJ, Wong ML, Bowden S, Liew CT, Hui AY, Wong VW, et al. Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy. Gastroenterology 2005;128:1890-1897. 11. Volz T, Lutgehetmann M, Wachtler P, Jacob A, Quaas A, Murray JM, et al. Impaired intrahepatic hepatitis B virus productivity contributes to low viremia in most HBeAg-negative patients. Gastroenterology 2007;133:843-852. 12. de la Fuente RA, Gutierrez ML, Garcia-Samaniego J, Fernandez-Rodriguez C, Lledo JL, Castellano G. Pathogenesis of occult chronic hepatitis B virus infection. World J Gastroenterol 2011;17:1543-1548. 13. Moolla N, Kew M, Arbuthnot P. Regulatory elements of hepatitis B virus transcription. J Viral Hepat 2002;9:323-331. 14. Guidotti LG, Rochford R, Chung J, Shapiro M, Purcell R, Chisari FV. Viral clearance without destruction of infected cells during acute HBV infection. Science 1999;284:825-829. 15. Gunther S, Sommer G, Von Breunig F, Iwanska A, Kalinina T, Sterneck M, et al. Amplification of full-length hepatitis B virus genomes from samples from patients with low levels of viremia: frequency and functional consequences of PCR-introduced mutations. J Clin Microbiol 1998;36:531-538. 16. Sheldon J, Rodes B, Zoulim F, Bartholomeusz A, Soriano V. Mutations affecting the replication capacity of the hepatitis B virus. J Viral Hepat 2006;13:427-434. 17. Jammeh S, Tavner F, Watson R, Thomas HC, Karayiannis P. Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol 2008;89:901-909. 18. Parekh S, Zoulim F, Ahn SH, Tsai A, Li J, Kawai S, et al. Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants. J Virol 2003;77:6601-6612. 19. Tong S. Mechanism of HBV genome variability and replication of HBV mutants. J Clin Virol 2005;34 Suppl 1:S134-138. 20. Pollicino T, Belloni L, Raffa G, Pediconi N, Squadrito G, Raimondo G, et al. Hepatitis B virus replication is regulated by the acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones. Gastroenterology 2006;130:823-837. 21. Belloni L, Pollicino T, De Nicola F, Guerrieri F, Raffa G, Fanciulli M, et al. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci U S A 2009;106:19975-19979. 11 2011년 대한간학회 추계 Single Topic Symposium 22. Miller RH, Robinson WS. Integrated hepatitis B virus DNA sequences specifying the major viral core polypeptide are methylated in PLC/PRF/5 cells. Proc Natl Acad Sci U S A 1983;80:2534-2538. 23. Korba BE, Wilson VL, Yoakum GH. Induction of hepatitis B virus core gene in human cells by cytosine demethylation in the promoter. Science 1985;228:1103-1106. 24. Bowyer SM, Dusheiko GM, Schoub BD, Kew MC. Expression of the hepatitis B virus genome in chronic hepatitis B carriers and patients with hepatocellular carcinoma. Proc Natl Acad Sci U S A 1987;84:847-850. 25. Araki K, Akagi K, Miyazaki J, Matsubara K, Yamamura K. Correlation of tissue-specific methylation with gene inactivity in hepatitis B virus transgenic mice.
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