Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels

Chunkyu Ko, Anindita Chakraborty, Wen Min Chou, Julia Hasreiter, Jochen M. Wettengel, Daniela Stadler, Romina Bester, Theresa Asen, Ke Zhang, Karin Wisskirchen, Jane A. McKeating, Wang-Shick Ryu, Ulrike Protzer

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Background & Aims: Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture. Methods: We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection. Results: HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5–12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days. Conclusions: After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents. Lay summary: Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.

Original languageEnglish
Pages (from-to)1231-1241
Number of pages11
JournalJournal of Hepatology
Volume69
Issue number6
DOIs
Publication statusPublished - 2018 Dec 1

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Recycling
Coinfection
Hepatitis B virus
Genome
Circular DNA
Infection
Virus Diseases
Life Cycle Stages
Taurocholic Acid
Capsid
Viral RNA
Viral Proteins
Nucleosides
Virion
Antiviral Agents
Half-Life
Clone Cells
Maintenance

All Science Journal Classification (ASJC) codes

  • Hepatology

Cite this

Ko, C., Chakraborty, A., Chou, W. M., Hasreiter, J., Wettengel, J. M., Stadler, D., ... Protzer, U. (2018). Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels. Journal of Hepatology, 69(6), 1231-1241. https://doi.org/10.1016/j.jhep.2018.08.012
Ko, Chunkyu ; Chakraborty, Anindita ; Chou, Wen Min ; Hasreiter, Julia ; Wettengel, Jochen M. ; Stadler, Daniela ; Bester, Romina ; Asen, Theresa ; Zhang, Ke ; Wisskirchen, Karin ; McKeating, Jane A. ; Ryu, Wang-Shick ; Protzer, Ulrike. / Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels. In: Journal of Hepatology. 2018 ; Vol. 69, No. 6. pp. 1231-1241.
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abstract = "Background & Aims: Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture. Methods: We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection. Results: HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5–12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days. Conclusions: After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents. Lay summary: Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of na{\"i}ve cells by newly formed virions.",
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Ko, C, Chakraborty, A, Chou, WM, Hasreiter, J, Wettengel, JM, Stadler, D, Bester, R, Asen, T, Zhang, K, Wisskirchen, K, McKeating, JA, Ryu, W-S & Protzer, U 2018, 'Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels', Journal of Hepatology, vol. 69, no. 6, pp. 1231-1241. https://doi.org/10.1016/j.jhep.2018.08.012

Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels. / Ko, Chunkyu; Chakraborty, Anindita; Chou, Wen Min; Hasreiter, Julia; Wettengel, Jochen M.; Stadler, Daniela; Bester, Romina; Asen, Theresa; Zhang, Ke; Wisskirchen, Karin; McKeating, Jane A.; Ryu, Wang-Shick; Protzer, Ulrike.

In: Journal of Hepatology, Vol. 69, No. 6, 01.12.2018, p. 1231-1241.

Research output: Contribution to journalArticle

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T1 - Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels

AU - Ko, Chunkyu

AU - Chakraborty, Anindita

AU - Chou, Wen Min

AU - Hasreiter, Julia

AU - Wettengel, Jochen M.

AU - Stadler, Daniela

AU - Bester, Romina

AU - Asen, Theresa

AU - Zhang, Ke

AU - Wisskirchen, Karin

AU - McKeating, Jane A.

AU - Ryu, Wang-Shick

AU - Protzer, Ulrike

PY - 2018/12/1

Y1 - 2018/12/1

N2 - Background & Aims: Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture. Methods: We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection. Results: HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5–12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days. Conclusions: After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents. Lay summary: Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.

AB - Background & Aims: Several steps in the HBV life cycle remain obscure because of a lack of robust in vitro infection models. These steps include particle entry, formation and maintenance of covalently closed circular (ccc) DNA, kinetics of gene expression and viral transmission routes. This study aimed to investigate infection kinetics and cccDNA dynamics during long-term culture. Methods: We selected a highly permissive HepG2-NTCP-K7 cell clone engineered to express sodium taurocholate co-transporting polypeptide (NTCP) that supports the full HBV life cycle. We characterized the replication kinetics and dynamics of HBV over six weeks of infection. Results: HBV infection kinetics showed a slow infection process. Nuclear cccDNA was only detected 24 h post-infection and increased until 3 days post-infection (dpi). Viral RNAs increased from 3 dpi reaching a plateau at 6 dpi. HBV protein levels followed similar kinetics with HBx levels reaching a plateau first. cccDNA levels modestly increased throughout the 45-day study period with 5–12 copies per infected cell. Newly produced relaxed circular DNA within capsids was reimported into the nucleus and replenished the cccDNA pool. In addition to intracellular recycling of HBV genomes, secondary de novo infection events resulted in cccDNA formation. Inhibition of relaxed circular DNA formation by nucleoside analogue treatment of infected cells enabled us to measure cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days. Conclusions: After a slow infection process, HBV maintains a stable cccDNA pool by intracellular recycling of HBV genomes and via secondary infection. Our results provide important insights into the dynamics of HBV infection and support the future design and evaluation of new antiviral agents. Lay summary: Using a unique hepatocellular model system designed to support viral growth, we demonstrate that hepatitis B virus (HBV) has remarkably slow infection kinetics. Establishment of the episomal transcription template and the persistent form of the virus, so called covalently closed circular DNA, as well as viral transcription and protein expression all take a long time. Once established, HBV maintains a stable pool of covalently closed circular DNA via intracellular recycling of HBV genomes and through infection of naïve cells by newly formed virions.

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