Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum

Stadelmayer, Bernd; Diederichs, Audrey; Chapus, Fleur; Rivoire, Michel; Neveu, Grégory; Alam, Antoine; Fraisse, Laurent; Carter, Kara; Testoni, Barbara; Zoulim, Fabien

doi:10.1016/j.jhep.2020.01.028

Cited by 63 publications

(91 citation statements)

References 78 publications

(90 reference statements)

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“…Consistent with previous studies [8,14,15,24], Northern blot assay confirmed that the serum/supernatant HBV RNA are predominantly short pgRNA species of heterogeneous lengths (Fig 5). Profiling of HBV RNA by RT-PCR further demonstrated that a substantial amount of spliced pgRNA variants exist in both the culture supernatant and patient serum samples.…”

Section: Plos Pathogenssupporting

confidence: 91%

“…Previous studies reported that the spliced pgRNAs also represent a species of extracellular HBV RNAs [14,15,24]. To assess whether the observed heterogeneous length of extracellular HBV RNA was due to RNA splicing ( Fig 5), the supernatant RNA from pHBV1.3-gtC-transfected HepG2 cells were reversed transcribed into cDNA by an HBV-specific RT primer, followed by PCR amplification with primers specifically targeting pcRNA, unspliced and spliced pgRNA, respectively ( Fig 6A).…”

Section: Plos Pathogensmentioning

confidence: 99%

“…Nonetheless, the current results suggest that the HBV RNA-virion does not essentially blur the distinction between para-retroviruses and conventional retroviruses. Future study should be carried out to specifically evaluate the infectivity of unspliced pgRNA-and spliced pgRNA-containing virions in vitro and in vivo, and to determine the biological functions of these RNA-virions, including the HBx mRNA-containing virions [15,40], in HBV lifecycle and virus-host interactions.…”

Section: Plos Pathogensmentioning

confidence: 99%

“…However, the nature of serum HBV RNA remains elusive. It has been reported that extracellular HBV RNA contains pgRNA and spliced RNA variants in the supernatant of HBV-infected cell cultures and in CHB patient serum [14,15]. Another study reported that the serum HBV RNA are of heterogeneous lengths, ranging from the full-length pgRNA to a few hundred nucleotides with 3' end truncations [8].…”

Section: Introductionmentioning

confidence: 99%

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Biogenesis and molecular characteristics of serum hepatitis B virus RNA

Shen

Xie

et al. 2020

PLoS Pathog

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HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent-and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope-knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3' RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3'-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.

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Section: Plos Pathogenssupporting

confidence: 91%

Section: Plos Pathogensmentioning

confidence: 99%

Section: Plos Pathogensmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biogenesis and molecular characteristics of serum hepatitis B virus RNA

Shen

Xie

et al. 2020

PLoS Pathog

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“…The HBV genome is transcribed by the host RNA polymerase II (RNA pol II) complex from unique promoters (basal core promoter (BCP), Sp1, Sp2 and Xp) and transcription start sites (7). This results in the generation of six major viral RNAs of increasing length with heterogeneous 5’ ends and a common polyadenylation signal (10, 11). The 3.5kb preC transcript encodes pre-core or e antigen (HBe) protein.…”

Section: Introductionmentioning

confidence: 99%

The CCCTC-binding factor CTCF represses hepatitis B virus Enhancer I and regulates viral transcription

D’Arienzo

Ferguson

Giraud

et al. 2020

Preprint

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26Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to 27 the virus during their lifetime and at risk of progressive liver disease, cirrhosis and 28 hepatocellular carcinoma. HBV is a member of the hepadnaviridae family that replicates via 29 episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of 30 this small viral genome, with overlapping open reading frames and regulatory elements, 31 suggests an important role for epigenetic pathways to regulate viral transcription. The 32 chromatin-organising transcriptional insulator protein CCCTC-binding factor (CTCF) has been 33 reported to regulate transcription in a diverse range of viruses. We identified two conserved 34 CTCF binding sites in the HBV genome within Enhancer I and chromatin immunoprecipitation 35 (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies 36 of the viral genome. siRNA knockdown of CTCF results in a significant increase in pre-genomic 37 RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA 38 replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF 39 binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in 40 repressing HBV transcription. 41 IMPORTANCE 42Hepatitis B virus (HBV) is a global cause of liver disease. At least 300 million individuals are 43 chronically infected with HBV, frequently leading to life-threatening liver cirrhosis and cancer. 44Following viral entry, HBV DNA enters the nucleus and is bound by histones that are subject to 45 epigenetic modification. The HBV genome contains two enhancer elements that stimulate viral 46 transcription but the interplay between the viral enhancers and promoters is not fully 47 understood. We have identified the host cell protein CCCTC binding factor (CTCF) as a 48 repressor of HBV gene expression. CTCF binds to the HBV genome within Enhancer I and 49 represses transcription of pre-genomic RNA. These findings provide new insights into how HBV 50 transcription is regulated and show a new role for CTCF as a transcriptional insulator by 51 associating with the viral genome between Enhancer I and the downstream basal core 52 promoter.53 54 55 4 (14). The BCP encodes a negative regulatory element (NRE) that overlaps with Enh II (15) and 87 has been reported to repress EnhII-mediated promoter activation (16). 88 Nuclear HBV cccDNA is assembled into nucleosomes by cellular histones to form episomal 89 chromatin (17, 18). The viral DNA is enriched with active epigenetic histone modifications 90 including trimethylation of lysine 4 (H3K4Me3) and acetylation of lysine 27 on histone 3 91 (H3K27Ac) but devoid of the repressive marks such as trimethylation of lysine 27 on histone 3 92 (H3K27Me3) (19, 20). The overlap of active histone marks with RNA pol II occupancy suggests 93 that viral transcription is regulated by epigenetic modification. In support of this, treating de 94 novo infected p...

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Circulating HBV RNA: From biology to clinical applications

et al. 2022

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Chronic HBV infection can hardly be cured due to the persistence of an intrahepatic pool of viral covalently closed circular DNA (cccDNA) transcription template, which is refractory to current antivirals. The direct analyses of cccDNA quantity and transcriptional activity require an invasive biopsy. Recently, circulating HBV RNA has been identified as a promising noninvasive surrogate marker of cccDNA and can be used for monitoring disease progression and predicting prognosis of patients with chronic HBV infection. To better understand this surrogate biomarker of cccDNA, we reviewed the current knowledge about the molecular characteristics and potential clinical applications of circulating HBV RNA. Specifically, we summarized the reported species and existing forms of circulating HBV RNA and discussed their biogenesis and the capacity of de novo infection by RNA virions. Moreover, we described the potential applications of circulating HBV RNA in different clinical scenarios, such as classifying the phases of chronic HBV infection, analyzing sustained on‐treatment and off‐treatment outcomes of treated patients, as well as predicting HCC development. Perspectives on future research of circulating HBV RNA were also proposed in this review.

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Full-length 5'RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum

Cited by 63 publications

References 78 publications

Biogenesis and molecular characteristics of serum hepatitis B virus RNA

Biogenesis and molecular characteristics of serum hepatitis B virus RNA

The CCCTC-binding factor CTCF represses hepatitis B virus Enhancer I and regulates viral transcription

Circulating HBV RNA: From biology to clinical applications

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