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...