HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon

Mutz, Pascal; Metz, Philippe; Lempp, Florian A.; Bender, Silke; B, Qu; Schöneweis, Katrin; Seitz, Stefan; Tu, Thomas; Restuccia, A.; Frankish, Jamie; Dächert, Christopher; Schusser, Benjamin; Koschny, Ronald; Polychronidis, Georgios; Schemmer, Peter; Hoffmann, Katja; Baumert, Thomas F.; Binder, Marco; Urban, Stephan; Bartenschlager, Ralf

doi:10.1053/j.gastro.2018.01.044

Cited by 130 publications

(114 citation statements)

References 74 publications

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“…reports (18,35), IFN-␣ slightly inhibited cccDNA formation and HBeAg secretion in a dose-dependent manner when administered at 1,000 IU/ml ( Fig. 10B and C).…”

Section: Kinetics Of Cccdna Establishment In In Vitro-infected Hepatomentioning

confidence: 88%

T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

Lempp

et al. 2018

J Virol

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Chronic infection with the human hepatitis B virus (HBV) is a major health problem. Virus persistence requires the establishment and maintenance of covalently closed circular DNA (cccDNA), the episomal virus template in the nucleus of infected hepatocytes. Compared to replicative DNA intermediates (relaxed circular DNA [rcDNA]), copy numbers of cccDNA in infected hepatocytes are low. Accordingly, accurate analyses of cccDNA require enrichment of nuclear fractions and Southern blotting or selective quantitative PCR (qPCR) methods allowing discrimination of cccDNA and rcDNA. In this report, we analyzed cccDNA-specific primer pairs for their ability to amplify cccDNA selectively. Using mixtures of defined forms of HBV and genomic DNA, we determined the potential of different nucleases for targeted digestion of the open/relaxed circular DNA forms in the absence and presence of genomic DNA without affecting cccDNA. We found that the combination of T5 exonuclease with a primer set amplifying an approximately 1-kb fragment permits reliable quantification of cccDNA without the requirement of prior nucleus enrichment or Hirt extraction. We tested this method in four different in vitro infection systems and quantified cccDNA copy numbers at increasing multiplicities of inoculated genome equivalents. We further analyzed the kinetics of cccDNA formation and the effect of drugs (interferon, entry inhibitors, and capsid inhibitors) on cccDNA. Our method allows reliable cccDNA quantification at early stages of infection in the presence of a high excess of input virus and replicative intermediates and is thereby suitable for drug screening and investigation of cccDNA formation and maintenance. IMPORTANCE cccDNA elimination is a major goal in future curative regimens for chronic HBV patients. However, PCR-based assays for cccDNA quantification show a principally constrained specificity when high levels of input virus or replicative intermediates are present. Here, we characterized T5 exonuclease as a suitable enzyme for mediumthroughput in vitro assays that preserves cccDNA but efficiently removes rcDNA prior to PCR-based quantification. We compared T5 exonuclease with the previously described exonuclease III and showed that both nucleases are suitable for reliable quantification of cccDNA by PCR. We substantiated the applicability of our method through examination of early cccDNA formation and stable accumulation in several in vitro infection models and analyzed cccDNA stability after administration of anti-HBV drugs. Our results support the use of T5 exonuclease for fast and convenient rcDNA removal, especially for early cccDNA quantification and rapid drug testing in in vitro studies. Citation Qu B, Ni Y, Lempp FA, Vondran FWR, Urban S. 2018. T5 exonuclease hydrolysis of hepatitis B virus replicative intermediates allows reliable quantification and fast drug efficacy testing of covalently closed circular DNA by PCR. J Virol 92:e01117-18. https://doi .

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“…reports (18,35), IFN-␣ slightly inhibited cccDNA formation and HBeAg secretion in a dose-dependent manner when administered at 1,000 IU/ml ( Fig. 10B and C).…”

Section: Kinetics Of Cccdna Establishment In In Vitro-infected Hepatomentioning

confidence: 88%

T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

Lempp

et al. 2018

J Virol

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“…Immunofluorescence was performed as described in [65]. For cholesterol staining, Filipin-III (250 µg/mL, Sigma-Aldrich) was used instead of a primary and secondary antibody.…”

Section: Immunofluorescence (If) Filipin Staining and Bright Field Mmentioning

confidence: 99%

Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors

Dächert

Gladilin

Binder

2019

Viruses

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Chronic Hepatitis C virus (HCV) infection still constitutes a major global health problem with almost half a million deaths per year. To date, the human hepatoma cell line Huh7 and its derivatives is the only cell line that robustly replicates HCV. However, even different subclones and passages of this single cell line exhibit tremendous differences in HCV replication efficiency. By comparative gene expression profiling using a multi-pronged correlation analysis across eight different Huh7 variants, we identified 34 candidate host factors possibly affecting HCV permissiveness. For seven of the candidates, we could show by knock-down studies their implication in HCV replication. Notably, for at least four of them, we furthermore found that overexpression boosted HCV replication in lowly permissive Huh7 cells, most prominently for the histone-binding transcriptional repressor THAP7 and the nuclear receptor NR0B2. For NR0B2, our results suggest a finely balanced expression optimum reached in highly permissive Huh7 cells, with even higher levels leading to a nearly complete breakdown of HCV replication, likely due to a dysregulation of bile acid and cholesterol metabolism. Our unbiased expression-profiling approach, hence, led to the identification of four host cellular genes that contribute to HCV permissiveness in Huh7 cells. These findings add to an improved understanding of the molecular underpinnings of the strict host cell tropism of HCV.

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“…The innate immune response is not sufficiently stimulated in the early stage of HBV infection to enable viral clearance. The findings in liver tissues of chimpanzees and CHB patients as well as experiments in vitro have shown that the endogenous IFNα and interferon‐stimulated genes (ISGs) were all not induced while HBV infection . Some research reported that the level of IFNα released by HBV‐infected PHH cells was even decreased and the expression of antiviral genes was hardly observed even in co‐culture with Kupffer cells (KC) …”

Section: Relationship Between Host and Hbvmentioning

confidence: 99%

“…The and interferon-stimulated genes (ISGs) were all not induced while HBV infection. 19,40 Some research reported that the level of IFNα released by HBV-infected PHH cells was even decreased and the expression of antiviral genes was hardly observed even in co-culture with Kupffer cells (KC). 41 HBV modulates immune cells function in order to favour its infection even its maintenance.…”

Section: Immune Cells and Cytokinesmentioning

confidence: 99%

Insights for clinical diagnostic indicators of virus and host in chronic hepatitis B infection

Guo

Zeng

et al. 2020

Journal of Viral Hepatitis

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Covalently closed circular DNA (cccDNA), which is stably present in the nucleus of hepatocytes, is an important indicator for evaluating antiviral efficacy. Since cccDNA quantification requires an invasive procedure, serum biological markers that can effectively reflect the transcriptional activity of intrahepatic virus and the efficacy of treatment are required. Here, from the aspects of virus and host, we outline the focus of clinical research of HBV in recent years, including HBV RNA, empty virus, hepatitis B core‐related antigen and changes in the immune response. We briefly discuss their significance in predicting disease activity and monitoring treatment response in chronic hepatitis B. On this basis, some issues worthy of attention in laboratory diagnosis are proposed.

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HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon

Abstract: In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response.

Cited by 130 publications

References 74 publications

T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

Gene Expression Profiling of Different Huh7 Variants Reveals Novel Hepatitis C Virus Host Factors

Insights for clinical diagnostic indicators of virus and host in chronic hepatitis B infection

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