Over 316 million people are living with chronic hepatitis B and as many as one quarter of people living with HIV have hepatitis B virus (HBV) co-infection. HIV-HBV co-infection increases levels of HBV replication, liver disease development, and the risk of hepatocellular carcinoma. The key to HBV persistence is nucleus- resident, long-lived covalently closed circular DNA (cccDNA) coding for all viral proteins. Nucleos(t)ide analogue therapy (NUC) effectively reduces HBV DNA in the serum by halting reverse transcription of pregenomic RNA (pgRNA). Recently, NUC therapy has been associated with cccDNA transcriptional silencing leading to reduced pgRNA levels in the liver and HBV RNA in the serum, however cccDNA quantities across the liver remain stable. Efforts to develop a cure for HBV focus on the removal or long term control of cccDNA activity, the latter yielding a functional cure. An alternative treatment to NUC is pegylated interferon-α (PEG- IFN), a potent antiviral therapy with comparatively greater rates of functional cure (7%), defined as a durable loss of serum HBV surface antigen (HBsAg). A study following participants before and after PEG-IFN found that treatment non-responders had elevated proportions of spliced HBV (spHBV) DNA from total HBV DNA, supporting a role of spHBV in modulating IFN responsiveness. There are 20 identified spHBV transcripts derived from pgRNA, a subset of which encode noncanonical HBV proteins. A limited number of studies have demonstrated that spHBV expression disrupts IFN response signaling and possibly alters cccDNA transcription. Given their putative role in modulating the host immune response and viral transcription, we propose to compare spHBV in HBV mono-infection and HIV-HBV co-infection. Combining use of a novel multiprobe multiplex droplet digital PCR with direct sequencing, we will characterize spHBV in HIV-HBV co- infection and HBV monoinfection in serum from individuals in the MACS/WIHS Combined Cohort Study. We expect an enrichment of spHBV RNA within total HBV RNA in co-infection compared to mono-infection. Additionally, we will model the decay of spHBV during NUC in HIV-HBV co-infection and compare spHBV expression in the same person’s liver and blood. Additionally, we will perform deep sequencing of hepatocytes with high vs low HBV transcription in an HIV-HBV coinfection ancillary study of the Hepatitis B Research Network, focusing on innate response pathway genes. We will then overexpress candidate spHBV that are likely to affect IFN responses in HepG2-NTCP cells. Conversely, we will use siRNA to knockdown candidate spHBV and measure IFN responses in an HBV infected HepG2-NTCP cells. Using this model in the context of HBV infection, we will measure the interplay between cccDNA transcription, spHBV expression, and innate signaling. This study will be the first characterization of spHBV in HIV-HBV co-infection and has the goal of identifying novel therapeutic targets specifically affecting cccDN...