Single-Nucleotide Resolution Mapping of Hepatitis B Virus Promoters in Infected Human Livers and Hepatocellular Carcinoma.
Hepatitis B virus (HBV) is a major cause of liver diseases, including hepatocellular carcinoma (HCC), and more than 650,000 people die annually due to HBV-associated liver failure. Extensive studies of individual promoters have revealed that heterogeneous RNA 5' ends contribute to the complexity of HBV transcriptome and proteome. Here, we provide a comprehensive map of HBV transcription start sites (TSSs) in human liver, HCC, and blood, as well as several experimental replication systems, at a single-nucleotide resolution. Using CAGE (cap analysis of gene expression) analysis of 16 HCC/nontumor liver pairs, we identify 17 robust TSSs, including a novel promoter for the X gene located in the middle of the gene body, which potentially produces a shorter X protein translated from the conserved second start codon, and two minor antisense transcripts that might represent viral noncoding RNAs. Interestingly, transcription profiles were similar in HCC and nontumor livers, although quantitative analysis revealed highly variable patterns of TSS usage among clinical samples, reflecting precise regulation of HBV transcription initiation at each promoter. Unlike the variety of TSSs found in liver and HCC, the vast majority of transcripts detected in HBV-positive blood samples are pregenomic RNA, most likely generated and released from liver. Our quantitative TSS mapping using the CAGE technology will allow better understanding of HBV transcriptional responses in further studies aimed at eradicating HBV in chronic carriers.


OBJECTIVE
Despite the availability of a safe and effective vaccine, HBV infection remains a global health problem, and current antiviral protocols are not able to eliminate the virus in chronic carriers. Previous studies of the regulation of HBV transcription have described four major promoters and two enhancers, but little is known about their activity in human livers and HCC. We deeply sequenced the HBV RNA 5' ends in clinical human samples and experimental models by using a new, sensitive and quantitative method termed cap analysis of gene expression (CAGE). Our data provide the first comprehensive map of global TSS distribution over the entire HBV genome in the human liver, validating already known promoters and identifying novel locations. Better knowledge of HBV transcriptional activity in the clinical setting has critical implications in the evaluation of therapeutic approaches that target HBV replication.
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan.
Kübra
K
Altinel
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan marie-annick.buendia@inserm.fr kosuke.hashimoto@riken.jp.
Kosuke
K
Hashimoto
Laboratoire de Pathogenèse des Virus de l'Hépatite B, Institut Pasteur, Paris, France.
Yu
Y
Wei
Hepacivirus et Immunité Innée, UMR CNRS 3569, Institut Pasteur, Paris, France.
Christine
C
Neuveut
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan.
Ishita
I
Gupta
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan.
Ana Maria
AM
Suzuki
INSERM, U1193, Paul-Brousse Hospital, Hepatobiliary Centre, Villejuif, France.|Université Paris Sud, Faculté de Médecine Le Kremlin Bicêtre, Villejuif, France.
Alexandre
A
Dos Santos
Hepacivirus et Immunité Innée, UMR CNRS 3569, Institut Pasteur, Paris, France.
Pierrick
P
Moreau
Laboratoire de Pathogenèse des Virus de l'Hépatite B, Institut Pasteur, Paris, France.
Tian
T
Xia
RIKEN Center for Life Science Technologies, Division of Bio-function Dynamics Imaging, Wako, Saitama, Japan.
Soichi
S
Kojima
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan.
Sachi
S
Kato
Department of Internal Medicine, Iwate Medical University, Iwate, Japan.
Yasuhiro
Y
Takikawa
Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.
Isao
I
Hidaka
Department of Gastroenterology/Internal Medicine, Gifu University Graduate School of Medicine, Gifu, Japan.
Masahito
M
Shimizu
Department of Laboratory Medicine, Jikei University School of Medicine, Tokyo, Japan.
Tomokazu
T
Matsuura
Research Center for Medical Science, Jikei University School of Medicine, Tokyo, Japan.
Akihito
A
Tsubota
Department of Clinical Laboratory Medicine, The University of Tokyo, Tokyo, Japan.
Hitoshi
H
Ikeda
Department of Gastroenterology and Hepatology Saitama Medical Center, Saitama Medical University, Saitama, Japan.
Sumiko
S
Nagoshi
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan.
Harukazu
H
Suzuki
Laboratoire de Pathogenèse des Virus de l'Hépatite B, Institut Pasteur, Paris, France.
Marie-Louise
ML
Michel
INSERM, U1193, Paul-Brousse Hospital, Hepatobiliary Centre, Villejuif, France.|Université Paris Sud, Faculté de Médecine Le Kremlin Bicêtre, Villejuif, France.|Assistance Publique-Hôpitaux de Paris, Pôle de Biologie Médicale, Paul-Brousse Hospital, Villejuif, France.
Didier
D
Samuel
INSERM, U1193, Paul-Brousse Hospital, Hepatobiliary Centre, Villejuif, France marie-annick.buendia@inserm.fr kosuke.hashimoto@riken.jp.|Université Paris Sud, Faculté de Médecine Le Kremlin Bicêtre, Villejuif, France.
Marie Annick
MA
Buendia
INSERM, U1193, Paul-Brousse Hospital, Hepatobiliary Centre, Villejuif, France.|Université Paris Sud, Faculté de Médecine Le Kremlin Bicêtre, Villejuif, France.|Assistance Publique-Hôpitaux de Paris, Pôle de Biologie Médicale, Paul-Brousse Hospital, Villejuif, France.
Jamila
J
Faivre
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, Japan.
Piero
P
Carninci
D000328:Adult; D000368:Aged; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000821:virology*; D002874:Chromosome Mapping; D005260:Female; D016679:Genome, Viral; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000235:genetics* / Q000472:pathogenicity; D019694:Hepatitis B, Chronic / Q000821:virology*; D006801:Humans; D008099:Liver / Q000821:virology; D008113:Liver Neoplasms / Q000821:virology*; D008297:Male; D051379:Mice; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D012315:RNA Caps / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D024363:Transcription Initiation Site; D059467:Transcriptome
D012315:RNA Caps; D012367:RNA, Viral
Nucleic Acids Res. 2013 Jan;41(Database issue):D566-70
23125365
Microbiol Mol Biol Rev. 2000 Mar;64(1):51-68
10704474
Science. 2005 Sep 2;309(5740):1559-63
16141072
Proc Natl Acad Sci U S A. 1986 Feb;83(3):566-70
3456153
J Hepatol. 2011 Nov;55(5):996-1003
21376091
Nature. 2014 Mar 27;507(7493):462-70
24670764
Methods Mol Biol. 2014;1164:67-85
24927836
Nucleic Acids Res. 2014 Jan;42(Database issue):D975-9
24297256
J Gen Virol. 2003 Jul;84(Pt 7):1907-13
12810886
J Virol. 1992 Jul;66(7):4382-9
1318408
Nat Genet. 2012 May 27;44(7):765-9
22634754
J Virol. 2013 May;87(10):5554-63
23468504
J Biol Chem. 1994 Sep 9;269(36):22593-8
8077209
J Virol. 1993 May;67(5):2559-65
8474161
Biochim Biophys Acta. 1999 Mar 30;1453(3):330-40
10101251
Intervirology. 1999;42(2-3):81-99
10516464
Gut. 2015 May;64(5):820-9
25021421
Intervirology. 2014;57(3-4):141-50
25034481
Antiviral Res. 2006 Nov;72(2):116-24
16780964
Nature. 2014 Mar 27;507(7493):455-61
24670763
Brief Bioinform. 2013 Mar;14(2):178-92
22517427
World J Gastroenterol. 2007 Jan 7;13(1):48-64
17206754
Nature. 2012 Sep 6;489(7414):57-74
22955616
Nature. 2014 Mar 20;507(7492):381-5
24531765
Hepatology. 2015 Jan;61(1):66-76
25132147
J Hepatol. 2010 Apr;52(4):594-604
20185200
Nat Genet. 2006 Jun;38(6):626-35
16645617
Sci Signal. 2012 Jan 03;5(205):ra1
22215732
World J Gastroenterol. 2007 Jan 7;13(1):14-21
17206751
BMC Bioinformatics. 2014 May 16;15:144
24884663
J Virol. 1995 Jun;69(6):3647-57
7745713
J Virol. 2015 Feb;89(4):2448-52
25505068
Genome Res. 2015 Dec;25(12):1812-24
26510915
J Virol. 2007 Nov;81(22):12472-84
17804499
Nat Protoc. 2012 Feb 23;7(3):542-61
22362160
Proc Natl Acad Sci U S A. 2015 Oct 20;112(42):E5715-24
26438841
Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3819-23
7731990
Gastroenterology. 2004 Jun;126(7):1750-8
15188170
Virology. 1984 Nov;139(1):53-63
6495659
Gastroenterology. 2012 May;142(6):1264-1273.e1
22537432
Genome Res. 2008 Jan;18(1):1-12
18032727
Antimicrob Agents Chemother. 1997 Aug;41(8):1715-20
9257747
Nat Genet. 2014 Jun;46(6):558-66
24777452
Bioinformatics. 2009 Jul 15;25(14):1754-60
19451168
World J Gastroenterol. 2014 Jan 14;20(2):425-35
24574711
J Hepatol. 2016 Oct;65(4):700-10
27245431
Gastroenterology. 2008 Jun;134(7):1890-9; quiz 2155
18424269
J Virol. 1987 Mar;61(3):904-11
3806799
Hepatology. 2007 May;45(5):1179-86
17465002