Detection of Low Copy Number Integrated Viral DNA Formed by <em>In Vitro</em> Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...

Heidelberg University Hospital

Ingham Institute

Chronic infection with the Hepatitis B Virus (HBV) is a major cause of liver-related morbidity and mortality. One peculiar observation in cells infected with HBV (or with closely‑related animal hepadnaviruses) is the presence of viral DNA integration in the host cell genome, despite this form being a replicative dead-end for the virus. The frequent finding of somatic integration of viral DNA suggests an evolutionary benefit for the virus; however, the mechanism of integration, its functions, and the clinical implications remain unknown. Here we review the current body of knowledge of HBV DNA integration, with particular focus on the molecular mechanisms and its clinical implications (including the possible consequences of replication-independent antigen expression and its possible role in hepatocellular carcinoma). HBV DNA integration is likely to influence HBV replication, persistence, and pathogenesis, and so deserves greater attention in future studies.

HBV DNA Integration: Molecular Mechanisms and Clinical Implications.

Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of  infection models that have detectable integration events. Here, we have established an  system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of  HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an  system to interrogate the mechanisms of HBV DNA integration.Hepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the  family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs is not clear. Here, we have developed and characterized an  system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we show that integration already occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation.

Hepatitis B virus DNA integration occurs early in the viral life cycle in an  infection model via NTCP-dependent uptake of enveloped virus particles.

While chronic infection with the human hepatitis B virus (HBV) and hepatitis delta virus (HDV) inflict major health burdens worldwide, current therapies cannot cure patients. One possible novel approach is blocking virus entry to prevent the establishment of infection in naïve hepatocytes. As HBV and HDV use identical viral envelope proteins and the same entry mechanisms, such a strategy would target both viruses. Entry inhibitors (e.g. neutralizing antibodies) have been relegated to the limited role of prophylaxis. However, recent clinical data and infection studies show that new viral entry inhibitors could play a major role in eliminating intrahepatic HBV/HDV genomes. We highlight the consequences on viral persistence after preventing virus entry and the therapeutic benefits entry inhibitors could achieve.

Virus entry and its inhibition to prevent and treat hepatitis B and hepatitis D virus infections.

Infection with the Hepatitis B Virus (HBV) is one of the strongest risk-factors for liver cancer (hepatocellular carcinoma, HCC). One of the reported drivers of HCC is the integration of HBV DNA into the host cell genome, which may induce pro-carcinogenic pathways. These reported pathways include: induction of chromosomal instability; generation of insertional mutagenesis in key cancer-associated genes; transcription of downstream cancer-associated cellular genes; and/or formation of a persistent source of viral protein expression (particularly HBV surface and X proteins). The contribution of each of these specific mechanisms towards carcinogenesis is currently unclear. Here, we review the current knowledge of specific sites of HBV DNA integration into the host genome, which sheds light on these mechanisms. We give an overview of previously-used methods to detect HBV DNA integration and the enrichment of integration events in specific functional and structural cellular genomic sites. Finally, we posit a theoretical model of HBV DNA integration during disease progression and highlight open questions in the field.

Cellular Genomic Sites of Hepatitis B Virus DNA Integration.

Hepatitis B virus (HBV) integration into the host cell genome occurs early on in infection and reportedly induces pro-oncogenic changes in hepatocytes that drive HCC initiation. However, it remains unclear when these changes occur during hepatocarcinogenesis. Extensive expansion of hepatocyte clones with a selective advantage was shown to occur prior to cancer formation during the HBeAg-seroconversion phase of chronic HBV infection. We hypothesized that since integrations occur during the early stages of infection, cell phenotype could be altered and induce a selection advantage (e.g., through insertional mutagenesis or cis-mediated activation of downstream genes). Here, we analyzed the enrichment of genomic and functional patterns in the cellular host sequence adjacent to HBV DNA integration events. We examined 717 unique integration events detected in patients who have and have not undergone HBeAg-seroconversion (n = 41) or in an in vitro model system. We also used an in silico model to control for detection biases. We showed that the sites of HBV DNA integration were distributed throughout the entire host genome without obvious enrichment of specific structural or functional genomic features in the adjacent cellular genome during HBeAg-seroconversion. Currently, this is the most comprehensive characterization of HBV DNA integration events prior to hepatocarcinogenesis. Our results suggest no significant selection for (or against) specific cellular sites of HBV DNA integration occur during the clonal expansion phase of chronic HBV infection. Thus, HBV DNA integration events likely represent passenger events rather than active drivers of liver cancer, which was previously suggested.

Thomas Tu

Department of Infectious Diseases,
Molecular Virology,
Department of Infectious Diseases, Molecular Virology

Heidelberg University Hospital

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Thomas Tu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Infectious Diseases,Molecular Virology,Department of Infectious Diseases, Molecular Virology

Heidelberg University Hospital

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Department of Infectious Diseases,
Molecular Virology,
Department of Infectious Diseases, Molecular Virology