1887

Abstract

Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25 % of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5′ truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome.

Funding
This study was supported by the:
  • Wellcome Trust (Award 220171/Z/20/Z)
    • Principle Award Recipient: MAzim Ansari
  • Wellcome Trust (Award 110110/Z/15/Z)
    • Principle Award Recipient: PhilippaClare Matthews
  • Wellcome Trust (Award 203141/A/16/Z)
    • Principle Award Recipient: PaoloPiazza
  • Chinese Academy of Medical Sciences (Award 2018-I2M-2-002)
    • Principle Award Recipient: JaneA McKeating
  • Medical Research Council (Award MR/R022011/1)
    • Principle Award Recipient: JaneA McKeating
  • Wellcome Trust (Award 200838/Z/16/Z)
    • Principle Award Recipient: JaneA McKeating
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001856
2023-05-17
2024-03-04
Loading full text...

Full text loading...

/deliver/fulltext/jgv/104/5/jgv001856.html?itemId=/content/journal/jgv/10.1099/jgv.0.001856&mimeType=html&fmt=ahah

References

  1. World Health Organisation Hepatitis B vaccines: WHO positions paper. WER 2017; 27:369–392
    [Google Scholar]
  2. McNaughton AL, Revill PA, Littlejohn M, Matthews PC, Ansari MA. Analysis of genomic-length HBV sequences to determine genotype and subgenotype reference sequences. J Gen Virol 2020; 101:271–283 [View Article] [PubMed]
    [Google Scholar]
  3. Yu H, Yuan Q, Ge S-X, Wang H-Y, Zhang Y-L et al. Molecular and phylogenetic analyses suggest an additional hepatitis B virus genotype “I.”. PLoS One 2010; 5:e9297 [View Article] [PubMed]
    [Google Scholar]
  4. Sozzi V, Walsh R, Littlejohn M, Colledge D, Jackson K et al. In vitro studies show that sequence variability contributes to marked variation in hepatitis B virus replication protein expression, and function observed across genotypes. J Virol 2016; 90:10054–10064 [View Article] [PubMed]
    [Google Scholar]
  5. McNaughton AL, D’Arienzo V, Ansari MA, Lumley SF, Littlejohn M et al. Insights from deep sequencing of the HBV genome-unique, tiny, and misunderstood. Gastroenterology 2019; 156:384–399 [View Article] [PubMed]
    [Google Scholar]
  6. Lythgoe KA, Lumley SF, Pellis L, McKeating JA, Matthews PC. Estimating hepatitis B virus cccDNA persistence in chronic infection. Virus Evol 2021; 7:veaa063 [View Article] [PubMed]
    [Google Scholar]
  7. Hong X, Kim ES, Guo H. Epigenetic regulation of hepatitis B virus covalently closed circular DNA: implications for epigenetic therapy against chronic hepatitis B. Hepatology 2017; 66:2066–2077 [View Article] [PubMed]
    [Google Scholar]
  8. Stadelmayer B, Diederichs A, Chapus F, Rivoire M, Neveu G et al. Full-length 5’RACE identifies all major HBV transcripts in HBV-infected hepatocytes and patient serum. J Hepatol 2020; 73:40–51 [View Article] [PubMed]
    [Google Scholar]
  9. Ito N, Nakashima K, Sun S, Ito M, Suzuki T. Cell type diversity in hepatitis B virus RNA splicing and Its regulation. Front Microbiol 2019; 10:207 [View Article] [PubMed]
    [Google Scholar]
  10. Lim CS, Sozzi V, Littlejohn M, Yuen LKW, Warner N et al. Quantitative analysis of the splice variants expressed by the major hepatitis B virus genotypes. Microb Genom 2021; 7:mgen000492 [View Article] [PubMed]
    [Google Scholar]
  11. Bayliss J, Lim L, Thompson AJV, Desmond P, Angus P et al. Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma. J Hepatol 2013; 59:1022–1028 [View Article] [PubMed]
    [Google Scholar]
  12. Ko C, Chakraborty A, Chou W-M, Hasreiter J, Wettengel JM et al. Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels. J Hepatol 2018; 69:1231–1241 [View Article] [PubMed]
    [Google Scholar]
  13. Tu T, Budzinska MA, Shackel NA, Urban S. HBV DNA integration: molecular mechanisms and clinical implications. Viruses 2017; 9:75 [View Article] [PubMed]
    [Google Scholar]
  14. Wooddell CI, Yuen M-F, Chan HL-Y, Gish RG, Locarnini SA et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci Transl Med, 2017; 9:eaan0241 [View Article] [PubMed]
    [Google Scholar]
  15. Magri A, Harris JM, D’Arienzo V, Minisini R, Jühling F et al. Inflammatory gene expression associates with hepatitis B virus cccDNA- but not integrant-derived transcripts in HBeAg negative disease. Viruses 2022; 14:1070 [View Article] [PubMed]
    [Google Scholar]
  16. Sells MA, Zelent AZ, Shvartsman M, Acs G. Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions. J Virol 1988; 62:2836–2844 [View Article] [PubMed]
    [Google Scholar]
  17. D’Arienzo V, Ferguson J, Giraud G, Chapus F, Harris JM et al. The CCCTC-binding factor CTCF represses hepatitis B virus enhancer I and regulates viral transcription. Cell Microbiol 2021; 23:e13274 [View Article] [PubMed]
    [Google Scholar]
  18. Prakash K, Larsson SB, Rydell GE, Andersson ME, Ringlander J et al. Hepatitis B Virus RNA profiles in liver biopsies by digital polymerase chain reaction. Hepatol Commun 2020; 4:973–982 [View Article] [PubMed]
    [Google Scholar]
  19. Ringlander J, Skoglund C, Prakash K, Andersson ME, Larsson SB et al. Deep sequencing of liver explant transcriptomes reveals extensive expression from integrated hepatitis B virus DNA. J Viral Hepat 2020; 27:1162–1170 [View Article] [PubMed]
    [Google Scholar]
  20. Astbury S, Costa Nunes Soares MM, Peprah E, King B, Jardim ACG et al. Nanopore sequencing from extraction-free direct PCR of dried serum spots for portable hepatitis B virus drug-resistance typing. J Clin Virol 2020; 129:104483 [View Article] [PubMed]
    [Google Scholar]
  21. Sauvage V, Boizeau L, Candotti D, Vandenbogaert M, Servant-Delmas A. Early MinION nanopore single-molecule sequencing technology enables the characterization 6 of hepatitis B virus genetic complexity in clinical samples. PLoS One 2018; 13:e0194366 [View Article] [PubMed]
    [Google Scholar]
  22. McNaughton AL, Roberts HE, Bonsall D, de Cesare M, Mokaya J et al. Illumina and nanopore methods for whole genome sequencing of hepatitis B virus (HBV). Sci Rep 2019; 9:7081 [View Article] [PubMed]
    [Google Scholar]
  23. Ramirez R, van Buuren N, Gamelin L, Soulette C, May L et al. Targeted long-read sequencing reveals comprehensive architecture, burden, and transcriptional signatures from hepatitis B virus-associated integrations and translocations in hepatocellular carcinoma cell lines. J Virol 2021; 95:e0029921 [View Article] [PubMed]
    [Google Scholar]
  24. Chow S, Kis O, Mulder DT, Danesh A, Bruce J et al. Myeloma immunoglobulin rearrangement and translocation detection through targeted capture sequencing. Life Sci Alliance 2023; 6:e202201543 [View Article] [PubMed]
    [Google Scholar]
  25. Shohdy KS, Bareja R, Sigouros M, Wilkes DC, Dorsaint P et al. Functional comparison of exome capture-based methods for transcriptomic profiling of formalin-fixed paraffin-embedded tumors. NPJ Genom Med 2021; 6:66 [View Article] [PubMed]
    [Google Scholar]
  26. Zhuo Z, Rong W, Li H, Li Y, Luo X et al. Long-read sequencing reveals the structural complexity of genomic integration of HBV DNA in hepatocellular carcinoma. NPJ Genom Med 2021; 6:84 [View Article] [PubMed]
    [Google Scholar]
  27. van Buuren N, Ramirez R, Soulette C, Suri V, Han D et al. Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B. JHEP Rep 2022; 4:100449 [View Article] [PubMed]
    [Google Scholar]
  28. Allweiss L, Strick-Marchand H. In-vitro and in-vivo models for hepatitis B cure research. Curr Opin HIV AIDS 2020; 15:173–179 [View Article] [PubMed]
    [Google Scholar]
  29. Ko C, Chakraborty A, Chou W-M, Hasreiter J, Wettengel JM et al. Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels. J Hepatol 2018; 69:1231–1241 [View Article] [PubMed]
    [Google Scholar]
  30. D’Arienzo V, Magri A, Harris JM, Wing PAC, Ko C et al. A PCR assay to quantify patterns of HBV transcription. J Gen Virol 2021; 102:001373 [View Article] [PubMed]
    [Google Scholar]
  31. Lumley SF, Jennings D, Waddilove E, Trebes A, Delphin M et al. Pan-genotypic probe-based enrichment to improve efficiency of hepatitis B virus sequencing. Genomics [View Article]
    [Google Scholar]
  32. Bonsall D, Ansari MA, Ip C, Trebes A, Brown A et al. ve-SEQ: robust, unbiased enrichment for streamlined detection and whole-genome sequencing of HCV and other highly diverse pathogens. F1000Res 2015; 4:1062 [View Article] [PubMed]
    [Google Scholar]
  33. Altinel K, Hashimoto K, Wei Y, Neuveut C, Gupta I et al. Single-nucleotide resolution mapping of hepatitis B Virus promoters in infected human livers and hepatocellular carcinoma. J Virol 2016; 90:10811–10822 [View Article] [PubMed]
    [Google Scholar]
  34. Lawrence M, Huber W, Pagès H, Aboyoun P, Carlson M et al. Software for computing and annotating genomic ranges. PLoS Comput Biol 2013; 9:e1003118 [View Article] [PubMed]
    [Google Scholar]
  35. Pagès H, Aboyoun P, Gentleman RSD. Biostrings: Efficient manipulation of biological strings; 2022 https://bioconductor.org/packages/Biostrings
  36. Law CW, Chen Y, Shi W, Smyth GK. Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol 2014; 15:R29 [View Article] [PubMed]
    [Google Scholar]
  37. Phipson B, Lee S, Majewski IJ, Alexander WS, Smyth GK. Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Ann Appl Stat 2016; 10:946–963 [View Article] [PubMed]
    [Google Scholar]
  38. Testoni B, Lebossé F, Scholtes C, Berby F, Miaglia C et al. Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients. J Hepatol 2019; 70:615–625 [View Article] [PubMed]
    [Google Scholar]
  39. Suslov A, Heim MH, Wieland S. Studying hepatitis virus-host interactions in patient liver biopsies. Viruses 2022; 14:2490 [View Article] [PubMed]
    [Google Scholar]
  40. Niu C, Livingston CM, Li L, Beran RK, Daffis S et al. The Smc5/6 complex restricts HBV when localized to ND10 without inducing an innate immune response and is counteracted by the HBV X protein shortly after infection. PLoS One 2017; 12:e0169648 [View Article] [PubMed]
    [Google Scholar]
  41. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 2015; 43:e47 [View Article] [PubMed]
    [Google Scholar]
  42. Okonechnikov K, Golosova O, Fursov M. UGENE team Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 2012; 28:1166–1167 [View Article] [PubMed]
    [Google Scholar]
  43. Ma C, Xu W, Yang Q, Liu W, Xiang Q et al. Osteopetrosis-associated transmembrane protein 1 recruits RNA exosome to restrict hepatitis B virus replication. J Virol 2020; 94:11 [View Article] [PubMed]
    [Google Scholar]
  44. Biswas S, Rust LN, Wettengel JM, Yusova S, Fischer M et al. Long-term hepatitis B virus infection of rhesus macaques requires suppression of host immunity. Nat Commun 2022; 13:2995 [View Article] [PubMed]
    [Google Scholar]
  45. Maslac O, Wagner J, Sozzi V, Mason H, Svarovskaia J et al. Secreted hepatitis B virus splice variants differ by HBV genotype and across phases of chronic hepatitis B infection. J Viral Hepat 2022; 29:604–615 [View Article] [PubMed]
    [Google Scholar]
  46. Eckmann CR, Rammelt C, Wahle E. Control of poly(A) tail length. Wiley Interdiscip Rev RNA 2011; 2:348–361 [View Article] [PubMed]
    [Google Scholar]
  47. Zhang G, Luo H, Li X, Hu Z, Wang Q. The dynamic poly(A) tail acts as a signal hub in mRNA metabolism. Cells 2023; 12:572 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001856
Loading
/content/journal/jgv/10.1099/jgv.0.001856
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error