Initial sites of hepadnavirus integration into host genome in human hepatocytes and in the woodchuck model of hepatitis B-associated hepatocellular carcinoma

R Chauhan,N D Churchill,T I Michalak,P M Mulrooney-Cousins

doi:10.1038/oncsis.2017.22

R Chauhan, N D Churchill + Show 2 more

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https://doi.org/10.1038/oncsis.2017.22

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Journal: Oncogenesis	Publication Date: Apr 1, 2017
Citations: 38	License type: open-access

Affiliation: Memorial University of Newfoundland

Abstract

Hepatitis B virus (HBV) and the closely related woodchuck hepatitis virus (WHV) are potent carcinogens that trigger development of primary hepatocellular carcinoma (HCC). The initial sites of hepadnavirus–host genome integration, their diversity and kinetics of formation can be central to virus persistence and the initiation and progression of HCC. To recognize the nature of the very early virus–host interactions, we explored de novo infection of human hepatocyte-like HepaRG cells with authentic HBV and naive woodchucks with WHV. HepaRG were analyzed from several minutes post exposure to HBV onwards, whereas woodchuck liver biopsies at 1 or 3 h and 6 weeks post infection with WHV. Inverse PCR and clonal sequencing of the amplicons were applied to identify virus–host genomic junctions. HBV and WHV DNA and their replication intermediates became detectable in one hour after virus exposure. Concomitantly, HBV DNA integration into various host genes was detected. Notably, junctions of HBV X gene with retrotransposon sequences, such as LINE1 and LINE2, became prominent shortly after infection. In woodchucks, insertion of WHV X and preS sequences into host genome was evident at 1 and 3 h post infection (h.p.i.), confirming that hepadnavirus under natural conditions integrates into hepatocyte DNA soon after invasion. The HBV and WHV X gene enhancer II/core promotor sequence most often formed initial junctions with host DNA. Moreover, multiple virus–virus DNA fusions appeared from 1 h.p.i. onwards in both infected hepatocytes and woodchuck livers. In summary, HBV DNA integrates almost immediately after infection with a variety of host’s sequences, among which tandemly repeating non-coding DNAs are common. This study revealed that HBV can engage mobile genetic elements from the beginning of infection to induce pro-oncogenic perturbations throughout the host genome. Such swift virus insertion was also evident in natural hepadnaviral infection in woodchucks.

Highlights

Hepatitis B virus (HBV) is a pro-oncogenic virus and chronic HBV infection is the main cause of primary hepatocellular carcinoma (HCC) with attributed 780 thousand deaths annually worldwide.[1,2] HBV carcinogenesis have been directly linked to the transforming properties of the virus X and truncated preS-S proteins and, in particular, to integration of viral DNA altering the host’s genome stability and expression of individual genes.[3,4,5] The integration of HBV X (HBx) and S gene sequences had been commonly observed in HBV-associated HCC and the prevailing opinion was that the virus insertions are random throughout the liver genome
Differentiated HepaRG cells were exposed to HBV in plasma from treatment-naive patients NL01.A, NL02.C and NL03.E with chronic hepatitis B (Table 1)
The cells became HBV DNA reactive from 1 h post infection (h.p.i.) at 1.8 × 103 ± 5.7 × 102 copies or virus genome equivalents/μg DNA

Summary

Introduction

Hepatitis B virus (HBV) is a pro-oncogenic virus and chronic HBV infection is the main cause of primary hepatocellular carcinoma (HCC) with attributed 780 thousand deaths annually worldwide.[1,2] HBV carcinogenesis have been directly linked to the transforming properties of the virus X and truncated preS-S proteins and, in particular, to integration of viral DNA altering the host’s genome stability and expression of individual genes.[3,4,5] The integration of HBV X (HBx) and S gene sequences had been commonly observed in HBV-associated HCC and the prevailing opinion was that the virus insertions are random throughout the liver genome. These chimeric transcripts display tumor promoting properties and may foster liver injury by sequestering hepatocellular microRNA-122.9,10 It might be that fusion of viral DNA with LINE1 may spread viral genetic material across the host’s genome owing to the intrinsic mobility of this retrotransposon.[11,12]

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