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Covalently closed circular DNA activity in HBV infection 분당서울대학교병원내과 김진욱 Covalently closed circular DNA (cccdna) is a viral minichromosome from which HBV RNA replication intermediate (pregenomic RNA)

1 Covalently closed circular DNA activity in HBV infection 분당서울대학교병원내과 김진욱 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) 는간세포핵내에존재하는 double-stranded circular DNA 로서, HBV 증식에필수적인 RNA intermediate인 pregenomic RNA (pgrna) 의 template로작용하며항바이러스약제중단시 HBV가다시증식하게하는주원인이다. 1 HBV cccdna 의정성및정량검사법 HBV cccdna를정제하기위하여우선 chromosomally integrated linear HBV DNA를제거할필요가있다. 이를위하여 SDS/NaCl을이용한 differential precipitation (Hirt method) 을통하여 protein-rich cellular DNA를침전시키고상층액에서 cccdna fraction을얻는다. 2 여기에 Plasmid-safe DNase 를처리하면 circular double stranded DNA만남기고 cellular DNA는선택적으로제거되어 genomic DNA의 contamination을재차차단한다. 하지만 Plasmid-safe DNase 로 relaxed circular DNA (rcdna) 를완전히제거할수는없으며, rcdna 에존재하는 gap을중간에포함하도록 primer를제작, 이용하는 cccdna-specific PCR로 cccdna를검출, 정량한다. 3,4 8

2 김진욱 Covalently closed circular DNA activity in HBV infection CHB 의자연경과중간세포내 HBV cccdna copy number 의변화 간조직검사검체에서 HBV cccdna를 real-time PCR로정량한연구에서 cccdna는 HBeAg + CHB에서 HBeAg - CHB에비하여더높으며 3,5 HBeAg - CHB와 inactive carrier간에는차이를보이지않았다. 3 흥미롭게도 HBsAg이소실된환자에서도미량이지만 cccdna는검출되었다. 3 혈청에서 HBsAg이검출되지않지만간조직에 HBV DNA가존재하는 occult hepatitis B virus infection (OBI) 은최근에그개념이정립되었으며 6 핵내잔존하는 cccdna가그원인으로생각된다. 7 항바이러스치료후간세포내 HBV cccdna copy number 의변화 Adefovir를 48주간치료후간세포에서 cccdna는유의하게감소함이보고되었다 (-0.80 log 10 copies/cell, 84% reduction) 3. Lamivudine과 entecavir를 1년간투여한환자를대상으로한연구에서도후간조직내 cccdna는감소소견을보였다. 4,8 그러나이러한연구는소수의환자를대상으로하였으며, cccdna가유의하게감소하지않았다는보고도있어 9 더많은수의환자를대상으로추가연구가필요하다. 한편, 치료종료시점에서간조직내 cccdna의양이 sustained virologic response를예측할수있다는연구가있다. 10 CHB 의자연경과중 HBV cccdna activity 의변화및그조절기전 간세포내 HBV DNA양의결정요인으로서 HBV cccdna copy number 이외에도 cccdna의 transcriptional activity가영향을줄가능성이있다. 그근거로서, 1) HBeAg + CHB의경우 cccdna와혈청 HBV DNA 사이양의상관관계를보이는반면 HBeAg - CHB의경우상관관계를보이지않으며 cccdna activity를반영하는 virion productivity index (cytoplasmic rcdna/cccdna) 가감소되어있는점, 11 2) 상술하였듯이 OBI에서간세포내에 HBV cccdna가존재하여, 어떤방식으로든 cccdna의 transcriptional activity가억제되고있음을시사함 12 등이다. 아직까지 cccdna의 transcriptional activity가조절되는기전은확립되지않았으나 host immune mediated factor와 viral factor가기여할것으로생각되며, viral factor는다시 genetic change와 epigenetic factor로구분하여생각해볼수있다 Host immune factor HBV pgrna의 transcription은 basal core promoter (BCP) 와 core upstream regulatory sequence (CURS) 및 enhancer II의조절을받으며, 많은 transcription factor가이들부위에결합하여조절기능을나타낸다. 13 9

3 2011 년대한간학회추계 Single Topic Symposium Host immunity가여러 transcriptional factor를조절함으로써 cccdna transcriptional activity에영향을줄가능성이충분히예상되며 intracellular cytokine애의한 non-cytolytic mechanism이 cccdna activity를조절함이 acute HBV infection model에서시사되었으나 14 CHB에서 noncytolytic control mechanism에대해서는아직별로알려진바가없다. 2. Genetic change 실험적으로 HBV sequence에 mutation을유도하면 HBV replication이억제됨 15 으로미루어자연적으로유발된돌연변이가 HBV cccdna activity를조절할가능성을생각해볼수있으나, 이후 in vitro study에 서 precore mutation (G1896A, G1899A) 은 viral replication 에유의한영향을보이지않았다. 16,17 BCP 의 double mutation (A1762T/G1764A) 이 HBV 증식에미치는영향에대한연구는 pgrna의 transcription이증가했다는보고와 18,19 유의한영향이없다는상충된결과를보였다 Epigenetic control DNA와결합하는 histone의 modification (i.e. acetylation) 과 DNA methylation은유전자의발현을조절하는가장중요한 epigenetic mechanism이다. HBV cccdna activity의 epigenetic control은비교적최근에알려지기시작했으며, cccdna-bound histone의 acetylation여부가 cccdna의 activity에영향을미침이알려졌으며 20 HBx 단백이 histone acetylation의조절인자이다. 21 한편, cellular DNA에삽입된 HBV DNA는메틸화가일어남이보고되었으며, 22 이메틸화된 HBV DNA는발현이억제됨이알려진바있는데, 최근 HBV cccdna에서도유사한메틸화가일어남이보고되었으며 27,28 메틸화된 HBV cccdna는 transcriptional activity가저하됨이알려졌다. 29 결 론 HBV cccdna의 activity가조절되는기전은아직충분히밝혀지지않았으며, 이에대한연구는 HBV replication에대한이해를증진시킬뿐아니라 HBV cccdna activity의조절을통한새로운기전의항바이러스치료법을개발하는중요한시발점이될수있을것이다. 참고문헌 1. Seeger C, Mason WS. Hepatitis B virus biology. Microbiol Mol Biol Rev 2000;64: Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 1967;26: Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, et al. Persistence of cccdna 10

4 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: Moolla N, Kew M, Arbuthnot P. Regulatory elements of hepatitis B virus transcription. J Viral Hepat 2002;9: 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: 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: 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: 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: 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: Tong S. Mechanism of HBV genome variability and replication of HBV mutants. J Clin Virol 2005;34 Suppl 1:S 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: 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:

5 2011 년대한간학회추계 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: 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: 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: Araki K, Akagi K, Miyazaki J, Matsubara K, Yamamura K. Correlation of tissue-specific methylation with gene inactivity in hepatitis B virus transgenic mice. Jpn J Cancer Res 1990;81: Pourcel C, Tiollais P, Farza H. Transcription of the S gene in transgenic mice is associated with hypomethylation at specific sites and with DNase I sensitivity. J Virol 1990;64: Vivekanandan P, Thomas D, Torbenson M. Methylation regulates hepatitis B viral protein expression. J Infect Dis 2009;199: Guo Y, Li Y, Mu S, Zhang J, Yan Z. Evidence that methylation of hepatitis B virus covalently closed circular DNA in liver tissues of patients with chronic hepatitis B modulates HBV replication. J Med Virol 2009;81: Kim JW, Lee SH, Park YS, Hwang JH, Jeong SH, Kim N, et al. Replicative Activity of Hepatitis B Virus Is Negatively Associated with Methylation of Covalently Closed Circular DNA in Advanced Hepatitis B Virus Infection. Intervirology

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untitled Korean J Gastroenterol Vol. 63 No. 6, 335-340 http://dx.doi.org/10.4166/kjg.2014.63.6.335 pissn 1598-9992 eissn 2233-6869 REVIEW ARTICLE 만성 B 형간염에서 B 형간염표면항원정량의임상적의의 연종은 고려대학교의과대학내과학교실 Clinical Significance