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Viral-host interactions: characterizing cis and trans interactions of integrated HBV DNA with the human genome
Dissertation   Open access

Viral-host interactions: characterizing cis and trans interactions of integrated HBV DNA with the human genome

Yih-Ping Su
Doctor of Philosophy (Ph.D.), Drexel University
Oct 2024
DOI:
https://doi.org/10.17918/00010797
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Abstract

Chromosome conformation capture Genomic interaction Hepatitis B virus Hi-C Long-read sequencing PacBio Hepatoma Genetics
Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide and a high percentage of HCC cases have been associated with chronic Hepatitis B virus (HBV) infection. Although the integration of HBV proviral DNA is not required for the virus to complete its lifecycle, integrated HBV DNA (iDNA) has been found soon after infection; is frequently reported in HBV-related HCC cases; and is often located adjacent to or within known oncogenes. We and our collaborators previously reported that ground glass hepatocytes (GGH), a likely clonal expansion of HBV infected hepatocytes that display a unique morphology consistent with expression of HBV surface antigen (HBsAg) that persist following successful antiviral treatment, harbor iDNA containing pre-S deletion mutations. These observations together suggest that iDNA could be responsible for the consistent expression and accumulation of HBsAg in GGH. Aside from the direct disruption iDNA introduces, we are interested in whether iDNA could alter the host 3D genomic architecture as other integrated viruses had demonstrated such an impact. Therefore, we hypothesize that cis interactions of the HBV DNA with the host chromosomes (i.e. HBV DNA integration) leads to trans (distant site) interactions that alter the 3D chromatin structure, which can contribute to drivers of liver carcinogenesis. To test our hypothesis, we utilized the high-throughput chromatin conformation capture (Hi-C) assay which has been commonly used to dissect 3D conformation of the targeted chromosomes. However, Hi-C was developed and designed for short-read sequencing platforms which makes distinguishing interactions associated specifically with HBV iDNA from other interactions challenging. Thus, we adapted and modified the standard Hi-C protocol, to make it compatible with the PacBio long-read sequencing platform while also making it specific to HBV iDNA. In brief, we accomplished this by replacing the restriction endonuclease used in conventional Hi-C with a more infrequent cutter, incorporated adaptor ligation and amplification, and adapted HBV-targeted hybridization capture for long DNA sequences. Utilizing all these modifications, we successfully adapted the Hi-C technology to long-read platforms providing novel action-at-a-distance information with regard HBV iDNA's noncovalent DNA interactions. These achievements provide for the generation of a more comprehensive view of the viral-host genomic interactions, not only in cell lines, but also for clinical specimens whereby it is now possible to shed light on multiple aspects of iDNA's role in liver carcinogenesis including activation of proto-oncogene as drivers of liver tumorigenesis by both cis and trans mechanisms.

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