Hepatitis B virus (HBV) DNA integration is not driven by viral proteins

T Tu; B Zehnder; M Levy; G Micali; L Tran; O Dabere; N Main; N Shackel; S Urban

doi:10.1055/s-0038-1677293

Zeitschrift für Gastroenterologie, Table of Contents

Z Gastroenterol 2019; 57(01): e92
DOI: 10.1055/s-0038-1677293

5. Viral Hepatitis, Immunology

Georg Thieme Verlag KG Stuttgart · New York

Hepatitis B virus (HBV) DNA integration is not driven by viral proteins

Authors

T Tu

¹University Hospital Heidelberg, Germany
B Zehnder

¹University Hospital Heidelberg, Germany
M Levy

²South Western Sydney Clinical School, University of New South Wales, Australia

³Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia
G Micali

³Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia

⁴Gastroenterology and Liver laboratory, Ingham Institute for Applied Medical Research, Sydney, Australia
L Tran

³Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia

⁴Gastroenterology and Liver laboratory, Ingham Institute for Applied Medical Research, Sydney, Australia
O Dabere

³Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia

⁴Gastroenterology and Liver laboratory, Ingham Institute for Applied Medical Research, Sydney, Australia
N Main

³Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia

⁴Gastroenterology and Liver laboratory, Ingham Institute for Applied Medical Research, Sydney, Australia
N Shackel

²South Western Sydney Clinical School, University of New South Wales, Australia

³Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney, New South Wales, Australia

⁴Gastroenterology and Liver laboratory, Ingham Institute for Applied Medical Research, Sydney, Australia
S Urban

¹University Hospital Heidelberg, Germany

⁵German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany

Abstract

Full Text

Integration of viral DNA into host cell genome occurs early in HBV infection. HBV DNA integrations arise when HBV double-stranded linear (dsl)DNA acts as a substrate for the repair of cellular DNA breaks. Intriguingly, HBV DNA integration has been detected in most patients with HBV infection, despite this form not being necessary for new virion production. Of clinical importance, HBV DNA integration may drive cancer and viral persistence (e.g. as a source of HBV immunomodulatory surface antigen). It remains unknown if HBV actively drives its integration or if it occurs passively through cellular DNA repair factors. Thus, we explored HBV DNA integration and its dependence on various viral factors.

First, Huh7-NTCP cells were infected with either wild-type or a core antigen-knockout HBV mutant. Integration was detected using inverse nested PCR. Both viruses integrated at the same rate, indicating that HBV DNA integration can occur without formation of de novo reverse-transcribed virus genomes. Next, we compared integration resulting from 1) HBV infection, 2) transfection of DNA extracted from cell culture-derived HBV, and 3) transfection of a linearized plasmid-derived 1-mer of HBV DNA. All groups integrated at similar rates, suggesting HBV integration does not depend on viral proteins or RNA in the input virus but only needs the presence of dslDNA. Supporting this, we found that HBV integration by serum-derived HBV containing ˜6% dslDNA was ˜5-fold lower compared to cell culture-derived HBV at ˜30% dslDNA. Interestingly, we found that circulating dslDNA increases from HBeAg-positive (average =˜10%) to HBeAg-negative (˜25%) phase in patient serum with the greatest levels observed in HCC patients (> 50%). This suggests that HBV DNA integration increases as liver disease progresses. Finally, we used a novel RT inverse nested PCR assay to show that HBV DNA integrations are transcriptionally-active in primary tumour and surrounding non-tumour tissues in HBV-infected patients (n = 12). Thus, HBV DNA integrations can indeed contribute to the expression of HBsAg in a true infection.

In summary, HBV dslDNA integrates into the cellular genome without the help of specific viral protein factors. Rather, it is likely to occur through cellular DNA repair mechanisms. As HBV integrations (unlike the replicative episomal viral DNA form) are not lost during regenerative cell mitosis, they form a stable reservoir for HBV antigen expression in an infected liver. Our studies are now being extended usingHBV inocula with specific mutations in circularisation signals to see if higher dslDNA levels lead to higher integration rates. If HBV integration does indeed drive persistence and resistance to clearance by the immune response as reported, then prevention of ongoing integration (e.g. by inhibiting virus entry) should be considered.