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

Abstract

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon. Video Link The video component of this article can be found at https://www.jove.com/video/58202/ , very few (1 to 2) copies of each integration are present in a given pool of cells infected in vitro. We have also found that integration mainly occurs during the initial infection of

De novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA

“…Here, we used Huh7-NTCP cells, which we have previously shown to be a suitable system for quantifying HBV DNA integration. 32 , 37 We confirmed that comparable levels of cccDNA are formed by WT or ΔHBc HBV in this system ( Fig. S3 ).…”

“…Integrated HBV DNA in in vitro infected cells was quantified by inverse nested PCR, as previously described. 31 , 32 …”

“…Primers encoding the stop codon and flanking complementary sequences up-and downstream the T67 codon were used to amplify two overlapping fragments of pHBV1. Integrated HBV DNA in in vitro infected cells was quantified by inverse nested PCR, as previously described [31,32].…”