# HBV cccDNA transcriptional silencing with and without HIV

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2024 · $730,270

## 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.
Previously, we applied single-cell methods to HBV/HIV co-infected liver tissues and demonstrated that cccDNA
is transcriptionally suppressed during NUCs, although the phenomenon is likely to be reversible. We now
propose to uncover the mechanism underlying cccDNA transcriptional silencing since irreversible
transcriptional silencing 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 during NUCs using single-cell laser capture microdissection and droplet digital PCR
(scLCM/ddPCR) to quantify a cccDNA transcriptional index (cccDNA TI). We will compare the cccDNA TI
between HBV mono- and HBV/HIV co-infection. In aim two we will characterize host genes that are
differentially expressed with regards to HBV RNAs and compare these between HBV mono- and HBV/HIV co-
infection. We will apply the 10X Visium spatial gene expression analysis (10x VSGEA) platform to liver tissues
with high vs. low cccDNA TI, interrogating HBV transcription using the same platform. Genes of interest (GOI)
identified by 10X VSGEA will be confirmed after enriching for cells with high vs. low cccDNA TI using
scLCM/ddPCR. We will prove GOI are involved in regulating HBV transcription in vitro studies using
CRISPR/cas9 knockout in HBV-infected NTCP-expressing HepG2 and primary human hepatocytes. We will
perform co-immunoprecipitation of GOI with HBV proteins to confirm mechanisms. In aim three we will
characterize epigenetic modifications of cccDNA in liver tissues already characterized with high vs low cccDNA
TI, comparing findings in HBV mono- and HBV/HIV co-infection. We will quantify CpG methylation, histone
modifications, and open chomatin in persons with high vs low cccDNA TI. In vitro, we will test the actions of
methyltransferases, histone acetyl transferases, and histone deacetylase inhibitors on cccDNA transcription
using an infection model of HBV. Our proposal fills knowledge gaps in understanding how to transform
reversible cccDNA transcriptional supp...

## Key facts

- **NIH application ID:** 10838179
- **Project number:** 1R01AI181659-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** ASHWIN BALAGOPAL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $730,270
- **Award type:** 1
- **Project period:** 2024-07-22 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10838179

## Citation

> US National Institutes of Health, RePORTER application 10838179, HBV cccDNA transcriptional silencing with and without HIV (1R01AI181659-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10838179. Licensed CC0.

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