Mechanisms of HBV cccDNA transcriptional regulation in persons with and without HIV

Johns Hopkins University, Baltimore MD

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Abstract

Hepatitis B virus (HBV) infects >300 million people chronically and is the leading cause of end-stage liver disease and hepatocellular carcinoma (HCC), resulting in ~1 million deaths annually. HIV is a common co- infection in people with chronic hepatitis B (CHB) and worsens HBV and liver disease outcomes. Liver disease is a leading cause of death among PWH in the era of antiretrovirals, mostly due to viral hepatitis. Antiviral treatment with nucleos(t)ide analogues (NUCs) suppresses plasma HBV DNA but does not eliminate the covalently closed circular DNA (cccDNA) HBV template that resides in every infected cell. NUC interruption leads to HBV DNA rebound and clinical hepatitis. Therefore, finding an HBV cure is of major importance for HBV mono- and HIV/HBV co-infection. In the first funding cycle, we applied single-cell methods to HBV/HIV co-infected liver tissues and demonstrated that cccDNA is transcriptionally suppressed during NUCs. We now propose to understand the mechanism underlying this finding since permanently exploiting this mechanism therapeutically can lead to a functional cure. We propose an intensive study of NUC-associated cccDNA transcriptional suppression comparing people with HBV/HIV co- and HBV mono-infection. In aim one, we will quantify intrahepatic viral DNA and RNA quantities in hepatocytes from HBV mono- and HBV/HIV co-infected individuals using single-cell laser capture microdissection and our novel droplet digital PCR assays. We will quantify and compare the cccDNA transcriptional index (cccDNA TI), a measure of cccDNA transcription, in HBV mono- and HBV/HIV co- infection. In aim two we will use novel techniques to test whether cccDNA transcriptional suppression is due to genetic or epigenetic modifications of HBV using HBV mono- and HBV/HIV co-infected liver tissues from aim one. We will use Cas9-Nanopore sequencing to examine epigenetic modifications of CpG dinucleotides in cccDNA. We will use the NovaSeq platform to perform next-generation sequencing of cccDNA to look for mutations that may be associated with reduced transcription and to quantify global cccDNA transcriptional suppression of canonical viral genes and splice variants. In aim three we will test the hypothesis that the host response contributes to cccDNA transcriptional suppression and compare this response between HBV mono- and HBV/HIV co-infection. We will apply the novel 10X Visium spatial gene expression analysis (10x VSGEA) platform to liver tissue from Aim one to compare hepatocyte host transcriptomes in tissues with high vs. low cccDNA TI, directly interrogating HBV transcription using the same platform. We will confirm genes identified with 10X VSGEA using Tecan RNA sequencing, enriching for cells with high vs. low cccDNA TI. We will conduct in vitro studies to prove that genes of interest are associated with cccDNA transcriptional suppression, focusing on innate immune genes and HIV-associated immune activation. Our proposal fills knowledge gaps in understanding how to transform reversible cccDNA transcriptional suppression to a functional cure.