Abstract
ABSTRACTHepatitis 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.IMPORTANCE 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.
Highlights
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
Searching for cap analysis of gene expression (CAGE) tags that did not match human sequences, we identified a total of 376,770 tags that were mapped on the HBV genome using 16 representative HBV sequences curated by HBVdb [25]
The expression levels of total HBV transcripts were highly variable in different samples from as low as TATA box binding protein (TBP) to as high as beta-actin (ACTB) (Fig. 1A)
Summary
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. HBV is the prototype member of the hepadnavirus family, characterized by a compact DNA genome replicating with its own reverse transcriptase from an RNA intermediate [4] This virus is classified into eight major genotypes with distinct geographic distributions [5, 6]. The HBV genome carries four open reading frames (ORFs), which encode seven different proteins, including three surface proteins, the core and e antigens, the polymerase, and the X transactivator The expression of these genes is regulated by four promoters and two enhancers, which direct the production of six distinct mRNAs (two 3.5-kb transcripts for the core and e antigens, the polymerase, and for pregenomic RNA (pgRNA), one 2.4-kb transcript for the large surface protein (LHBs), two 2.1-kb transcripts for the middle and small surface proteins (MHBs and SHBs, respectively), and one 0.7-kb mRNA for the X transactiva-.
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