Hepatitis B Virus DNA Integration: In Vitro Models for Investigating Viral Pathogenesis and Persistence

Abstract

Hepatitis B Virus (HBV) is a globally-distributed pathogen and is a major cause of liver disease. HBV (or closely-related animal hepadnaviruses) can integrate into the host genome, but (unlike retroviruses) this integrated form is replication-defective. The specific role(s) of the integrated HBV DNA has been a long-standing topic of debate. Novel in vitro models of HBV infection combined with sensitive molecular assays now enable researchers to investigate this under-characterised phenomenon with greater ease and precision. This review covers the contributions these systems have made to understanding how HBV DNA integration induces liver cancer and facilitates viral persistence. We summarise the current findings into a working model of chronic HBV infection and discuss the clinical implications of this hypothetical framework on the upcoming therapeutic strategies used to curb HBV-associated pathogenesis.

Keywords: HBV DNA integration; HBV double-stranded linear DNA; hepatitis B virus; hepatocellular carcinoma (HCC); microhomology-mediated end joining; non-homologous end joining; viral persistence.

Figure 1

The viral RNAs and open reading frames (ORFs) expressed from the (A) cccDNA and (B) dslDNA forms of hepatitis B virus (HBV). The double red lines represent of the HBV DNA genome, blue lines represent viral mRNAs, triangles indicate transcriptional promotors, and coloured arrows represent viral ORFs. Nucleotide numbering of ORFs are shown as per Genbank Accession #AB241115. The viral mRNAs expressed from cccDNA terminate at a polyadenylation signal (poly A) located in the Core ORF. However, in dslDNA form, the canonical poly A signal is located at the 5′ end and mRNAs instead terminate at a non-canonical cryptic poly A signal. The mRNA coding for the HBV PreC/Core and Polymerase are separated from its promoter in the dslDNA form, leading to loss of their expression (dashed line). Figure generated using BioRender (https://biorender.com/).

Figure 2

Replication cycle of hepatitis B virus (HBV) including integration into host genome. HBV virions attach to heparan sulphate proteoglycans (HSPG) on the cell surface, allowing for clathrin-mediated endocytosis via sodium taurochlorate co-transporting polypeptide (NTCP). Relaxed circular (rc)DNA genomes (top) are converted into covalently closed circular (ccc)DNA, which serve as the transcriptional template for viral mRNAs (vRNAs) and pregenomic (pg)RNA. Double stranded linear (dsl)DNA (bottom) can also form cccDNA but cannot code for functional rcDNA due to an additional 16nt insertion (asterisk) [20,21,22]. HBV pgRNA is encapsidated by viral capsid proteins and is reverse transcribed by the viral polymerase to produce either rcDNA or dslDNA. The mature nucleocapsids containing viral DNA are then enveloped by HBsAg embedded into host membranes and secreted as virions. As a secondary pathway, dslDNA can integrate into the host genome at double stranded DNA breaks, via non-homologous end joining. Integrated HBV DNA is replication-deficient due to rearrangements of the viral genome that abrogate expression of the viral polymerase and capsid proteins (Figure 1). Figure adapted from [23] and generated using BioRender (https://biorender.com/).

Figure 3

A hypothetical model of host-virus dynamics over the course of a chronic HBV infection. The majority of HBsAg is derived from cccDNA immediately after initial infection, as HBV DNA integrations are rare. When activated, the immune response preferentially targets cells expressing HBeAg and Polymerase, which are coded by cccDNA but not integrated DNA. Loss of cccDNA-containing hepatocytes also leads to clearance of cccDNA from surrounding infected hepatocytes via mitosis. Daughter cells are susceptible to new reinfection, inducing new integration events. This cycle of clearance and reinfection continues, resulting in fluctuating levels of HBeAg and cccDNA as well as serum viral load. Meanwhile, selective clonal expansion of hepatocytes with integrated HBV DNA is driven by the lack of HBeAg and polymerase expression, as well as the expression of HBsAg (rendering them refractory to new infection due to super-infection exclusion). As integrated HBV DNA frequency increases, this results in reduced HBeAg and serum viral load, while HBsAg levels remain relatively stable. Figure was generated using BioRender (https://biorender.com/).