Skip to content
1887

Journal of General Virology header logo Journal of General Virology

Volume 106, Issue 3

NEDD4-binding protein 1 suppresses hepatitis B virus replication by regulating viral RNAs Open Access

Abstract

Chronic infection with hepatitis B virus (HBV) (chronic HBV infection) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogues are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug-resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We tested the effect of 132 RBPs on HBV replication by ectopically expressing these proteins along with HBV in hepatocellular carcinoma and evaluated the intracellular capsid-associated HBV DNA level. Our screen identified NEDD4-binding protein 1 (N4BP1) as having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, the overexpression of N4BP1 decreased core-associated HBV DNA levels, while knockdown or knockout of the gene encoding N4BP1 rescued core-associated HBV DNA levels. N4BP1 possesses the KH-like and RNase domains, and both were required for the anti-HBV effect of N4BP1. Additionally, we measured levels of HBV pregenomic RNA (pgRNA) and covalently closed circular DNA in the RBP-transfected cells and confirmed that N4BP1 binds pgRNA directly and regulates both the 3.5 and 2.4/2.1 kb HBV RNA. In summary, N4BP1 is a newly identified host factor able to counteract HBV production by regulating 3.5 and 2.1/2.4 kb HBV RNA.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antiviral , hepatitis B virus , NEDD4-binding protein 1 (N4BP1) , pregenomic RNA and RNA-binding protein
Funding
This study was supported by the:
  • Japan Society for the Promotion of Science (Award JP23K20041)
    • Principal Award Recipient: TakasukeFukuhara
  • Japan Agency for Medical Research and Development (Award JP22gm1610008)
    • Principal Award Recipient: TakasukeFukuhara
  • Japan Agency for Medical Research and Development (Award 22fk0310523h0001)
    • Principal Award Recipient: TakasukeFukuhara
  • Japan Agency for Medical Research and Development (Award 22fk0310524h0001)
    • Principal Award Recipient: TakasukeFukuhara
  • Japan Agency for Medical Research and Development (Award 22fk0310513h0001)
    • Principal Award Recipient: TakasukeFukuhara
  • Japan Agency for Medical Research and Development (Award 22fk0310506h0001)
    • Principal Award Recipient: TakasukeFukuhara
  • Japan Agency for Medical Research and Development (Award 22fk0310501h0001)
    • Principal Award Recipient: TakasukeFukuhara
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. The Microbiology Society waived the open access fees for this article.
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.002082
2025-03-20
2025-12-15

Metrics

Loading full text...

Full text loading...

/deliver/fulltext/jgv/106/3/jgv002082.html?itemId=/content/journal/jgv/10.1099/jgv.0.002082&mimeType=html&fmt=ahah

References

  1. World Health Organization Hepatitis B Fact Sheet; 2021 https://www.who.int/en/news-room/fact-sheets/detail/hepatitis-b
  2. World Health Organization Immunization Coverage; 2021 https://www.who.int/news-room/fact-sheets/detail/immunization-coverage
  3. Königer C, Wingert I, Marsmann M, Rösler C, Beck J et al. Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses. Proc Natl Acad Sci USA 2014; 111:E4244–53 [View Article] [PubMed]
     [Google Scholar]
  4. Qi Y, Gao Z, Xu G, Peng B, Liu C et al. DNA polymerase kappa is a key cellular factor for the formation of covalently closed circular DNA of hepatitis B Virus. PLoS Pathog 2016; 12:e1005893 [View Article] [PubMed]
     [Google Scholar]
  5. Sheraz M, Cheng J, Tang L, Chang J, Guo JT. Cellular DNA topoisomerases are required for the synthesis of hepatitis B virus covalently closed circular DNA. J Virol 2019; 93:e02230-18 [View Article] [PubMed]
     [Google Scholar]
  6. Kitamura K, Que L, Shimadu M, Koura M, Ishihara Y et al. Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus. PLoS Pathog 2018; 14:e1007124 [View Article] [PubMed]
     [Google Scholar]
  7. Rijckborst V, Janssen HLA. The role of interferon in hepatitis B therapy. Curr Hepat Rep 2010; 9:231–238 [View Article] [PubMed]
     [Google Scholar]
  8. Doong SL, Tsai CH, Schinazi RF, Liotta DC, Cheng YC. Inhibition of the replication of hepatitis B virus in vitro by 2’,3’-dideoxy-3’-thiacytidine and related analogues. Proc Natl Acad Sci USA 1991; 88:8495–8499 [View Article] [PubMed]
     [Google Scholar]
  9. Li Q, Sun B, Zhuo Y, Jiang Z, Li R et al. Interferon and interferon-stimulated genes in HBV treatment. Front Immunol 2022; 13:1034968 [View Article]
     [Google Scholar]
  10. Tang LSY, Covert E, Wilson E, Kottilil S. Chronic hepatitis B infection. JAMA 2018; 319:1802 [View Article]
     [Google Scholar]
  11. Dienstag JL, Perrillo RP, Schiff ER, Bartholomew M, Vicary C et al. A preliminary trial of lamivudine for chronic hepatitis B infection. N Engl J Med 1995; 333:1657–1661 [View Article] [PubMed]
     [Google Scholar]
  12. Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC et al. Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci USA 1996; 93:4398–4402 [View Article] [PubMed]
     [Google Scholar]
  13. Suzuki F, Akuta N, Suzuki Y, Sezaki H, Arase Y et al. Clinical and virological features of non-breakthrough and severe exacerbation due to lamivudine-resistant hepatitis B virus mutants. J Med Virol 2006; 78:341–352 [View Article]
     [Google Scholar]
  14. Mao R, Nie H, Cai D, Zhang J, Liu H et al. Inhibition of hepatitis B virus replication by the host zinc finger antiviral protein. PLoS Pathog 2013; 9:e1003494 [View Article] [PubMed]
     [Google Scholar]
  15. Li M, Yang J, Zhao Y, Song Y, Yin S et al. MCPIP1 inhibits Hepatitis B virus replication by destabilizing viral RNA and negatively regulates the virus-induced innate inflammatory responses. Antiviral Res 2020; 174:104705 [View Article] [PubMed]
     [Google Scholar]
  16. Sun S, Nakashima K, Ito M, Li Y, Chida T et al. Involvement of PUF60 in transcriptional and post-transcriptional regulation of hepatitis B virus pregenomic RNA expression. Sci Rep 2017; 7:12874 [View Article]
     [Google Scholar]
  17. Nakai M, Komiya K, Murata M, Kimura T, Kanaoka M et al. Expression of pIX gene induced by transgene promoter: possible cause of host immune response in first-generation adenoviral vectors. Hum Gene Ther 2007; 18:925–936 [View Article] [PubMed]
     [Google Scholar]
  18. Suzuki M, Kondo S, Pei Z, Maekawa A, Saito I et al. Preferable sites and orientations of transgene inserted in the adenovirus vector genome: the E3 site may be unfavorable for transgene position. Gene Ther 2015; 22:421–429 [View Article] [PubMed]
     [Google Scholar]
  19. Pei Z, Kondo S, Kanegae Y, Saito I. Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: application to vector titration. Biochem Biophys Res Commun 2012; 417:945–950 [View Article] [PubMed]
     [Google Scholar]
  20. Watashi K, Liang G, Iwamoto M, Marusawa H, Uchida N et al. Interleukin-1 and tumor necrosis factor-α trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID). J Biol Chem 2013; 288:31715–31727 [View Article] [PubMed]
     [Google Scholar]
  21. Yamasoba D, Sato K, Ichinose T, Imamura T, Koepke L et al. N4BP1 restricts HIV-1 and its inactivation by MALT1 promotes viral reactivation. Nat Microbiol 2019; 4:1532–1544 [View Article] [PubMed]
     [Google Scholar]
  22. Yamashita A, Tamaki M, Kasai H, Tanaka T, Otoguro T et al. Inhibitory effects of metachromin A on hepatitis B virus production via impairment of the viral promoter activity. Antiviral Res 2017; 145:136–145 [View Article] [PubMed]
     [Google Scholar]
  23. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; 15:550 [View Article] [PubMed]
     [Google Scholar]
  24. Ogata H, Goto S, Sato K, Fujibuchi W, Bono H et al. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 1999; 27:29–34 [View Article] [PubMed]
     [Google Scholar]
  25. Wu T, Hu E, Xu S, Chen M, Guo P et al. clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation 2021; 2:100141 [View Article] [PubMed]
     [Google Scholar]
  26. Wickham H. ggplot2: Elegant Graphics for Data Analysis Springer-Verlag New York; 2016 [View Article]
     [Google Scholar]
  27. Narmada BC, Khakpoor A, Shirgaonkar N, Narayanan S, Aw PPK et al. Single-cell landscape of functionally cured chronic hepatitis B patients reveals activation of innate and altered CD4-CTL-driven adaptive immunity. J Hepatol 2024; 81:42–61 [View Article] [PubMed]
     [Google Scholar]
  28. Lampertico P, Agarwal K, Berg T, Buti M, Janssen HLA et al. EASL 2017 clinical practice guidelines on the management of hepatitis B virus infection. J Hepatol 2017; 67:370–398 [View Article]
     [Google Scholar]
  29. Zheng GXY, Terry JM, Belgrader P, Ryvkin P, Bent ZW et al. Massively parallel digital transcriptional profiling of single cells. Nat Commun 2017; 8:14049 [View Article] [PubMed]
     [Google Scholar]
  30. Wolf FA, Angerer P, Theis FJ. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 2018; 19:15 [View Article] [PubMed]
     [Google Scholar]
  31. Hao Y, Hao S, Andersen-Nissen E, Mauck WM 3rd, Zheng S et al. Integrated analysis of multimodal single-cell data. Cell 2021; 184:3573–3587 [View Article] [PubMed]
     [Google Scholar]
  32. Tang H, Huang Y, Chen J, Yu C, Huang AL. Cellular protein TIA-1 regulates the expression of HBV surface antigen by binding the HBV posttranscriptional regulatory element. Intervirol 2008; 51:203–209 [View Article] [PubMed]
     [Google Scholar]
  33. Xu W, Ma C, Zhang Q, Zhao R, Hu D et al. PJA1 coordinates with the SMC5/6 complex to restrict DNA viruses and episomal genes in an interferon-independent manner. J Virol 2018; 92:e00825-18 [View Article] [PubMed]
     [Google Scholar]
  34. Cai J, Yang L, Wang B, Huang Y, Tang J et al. Identification of a novel N4BP1-like gene from grass carp (Ctenopharyngodon idella) in response to GCRV infection. Fish Shellfish Immunol 2014; 36:223–228 [View Article] [PubMed]
     [Google Scholar]
  35. Iwamoto M, Watashi K, Tsukuda S, Aly HH, Fukasawa M et al. Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP. Biochem Biophys Res Commun 2014; 443:808–813 [View Article] [PubMed]
     [Google Scholar]
  36. König A, Yang J, Jo E, Park KHP, Kim H et al. Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells. J Hepatol 2019; 71:289–300 [View Article] [PubMed]
     [Google Scholar]
  37. Honkoop P, Niesters HG, de Man RA, Osterhaus AD, Schalm SW. Lamivudine resistance in immunocompetent chronic hepatitis B. Incidence and patterns. J Hepatol 1997; 26:1393–1395 [View Article] [PubMed]
     [Google Scholar]
  38. Tipples GA, Ma MM, Fischer KP, Bain VG, Kneteman NM et al. Mutation in HBV RNA-dependent DNA polymerase confers resistance to lamivudine in vivo. Hepatology 1996; 24:714–717 [View Article] [PubMed]
     [Google Scholar]
  39. Bartholomew MM, Jansen RW, Jeffers LJ, Reddy KR, Johnson LC et al. Hepatitis-B-virus resistance to lamivudine given for recurrent infection after orthotopic liver transplantation. Lancet 1997; 349:20–22 [View Article] [PubMed]
     [Google Scholar]
  40. Valverde R, Edwards L, Regan L. Structure and function of KH domains. FEBS J 2008; 275:2712–2726 [View Article] [PubMed]
     [Google Scholar]
  41. Hollingworth D, Candel AM, Nicastro G, Martin SR, Briata P et al. KH domains with impaired nucleic acid binding as a tool for functional analysis. Nucleic Acids Res 2012; 40:6873–6886 [View Article]
     [Google Scholar]
  42. Anantharaman V, Aravind L. The NYN domains: novel predicted RNAses with a PIN domain-like fold. RNA Biol 2006; 3:18–27 [View Article] [PubMed]
     [Google Scholar]
  43. Shi H, Sun L, Wang Y, Liu A, Zhan X et al. N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization. Nat Commun 2021; 12:1379 [View Article]
     [Google Scholar]
  44. Gitlin AD, Heger K, Schubert AF, Reja R, Yan D et al. Integration of innate immune signalling by caspase-8 cleavage of N4BP1. Nature 2020; 587:275–280 [View Article]
     [Google Scholar]
  45. Cui X, Li Y, Xu H, Sun Y, Jiang S et al. Characteristics of hepatitis B virus integration and mechanism of inducing chromosome translocation. NPJ Genom Med 2023; 8:11 [View Article] [PubMed]
     [Google Scholar]
  46. Gu H, Zheng S, Han G, Yang H, Deng Z et al. Porcine reproductive and respiratory syndrome virus adapts antiviral innate immunity via manipulating MALT1. mBio 2022; 13:e0066422 [View Article] [PubMed]
     [Google Scholar]
  47. Mino T, Takeuchi O. Regnase-1-related endoribonucleases in health and immunological diseases. Immunol Rev 2021; 304:97–110 [View Article] [PubMed]
     [Google Scholar]
  48. Melén K, Keskinen P, Lehtonen A, Julkunen I. Interferon-induced gene expression and signaling in human hepatoma cell lines. J Hepatol 2000; 33:764–772 [View Article] [PubMed]
     [Google Scholar]
  49. Marozin S, Altomonte J, Stadler F, Thasler WE, Schmid RM et al. Inhibition of the IFN-β response in hepatocellular carcinoma by alternative spliced isoform of IFN regulatory factor-3. Mol Ther 2008; 16:1789–1797 [View Article] [PubMed]
     [Google Scholar]
  50. Speer SD, Li Z, Buta S, Payelle-Brogard B, Qian L et al. ISG15 deficiency and increased viral resistance in humans but not mice. Nat Commun 2016; 7:11496 [View Article] [PubMed]
     [Google Scholar]
  51. Dickensheets H, Sheikh F, Park O, Gao B, Donnelly RP. Interferon-lambda (IFN-λ) induces signal transduction and gene expression in human hepatocytes, but not in lymphocytes or monocytes. J Leukoc Biol 2013; 93:377–385 [View Article] [PubMed]
     [Google Scholar]
/content/journal/jgv/10.1099/jgv.0.002082
Loading
NEDD4-binding protein 1 suppresses hepatitis B virus replication by regulating viral RNAs
J. Gen. Virol. 106, 002082 (2025); https://doi.org/10.1099/jgv.0.002082
/content/journal/jgv/10.1099/jgv.0.002082
/content/journal/jgv/10.1099/jgv.0.002082
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

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