Project Summary/Abstract Chronic infection by hepatitis B virus (HBV) is a leading cause of liver cancer worldwide, which can be promoted by hepatitis B e antigen (HBeAg) through induction of immune tolerance. Since HBeAg loss is a therapeutic goal, it is critically important to identify the host enzyme responsible for its production. While the structurally related core protein (p21; 183aa) assembles into capsids to provide the venue for genome replication, HBeAg is a secreted soluble protein. It is initially translated as fused precore/core protein (p25; 29+183aa), with the N-terminal 19aa targeting the protein to the secretory pathway followed by its cleavage. The resultant p22 is further cleaved at the C-terminus in the trans-Golgi network (TGN) to generate mature HBeAg. HBeAg production in cell lines can be blocked by a proprotein convertase (PC) inhibitor. PCs present in the TGN include furin, PACE4 and PC7, with most PCs preferring polybasic sequence while furin capable of cleaving after RXXR sequence. Four such motifs are present at the C-terminus of p22, with fused motifs 1 and 2 (151RRGRSPR157) and polybasic motifs 3 and 4 (RRRR). Previous mutational analysis identified HBeAg as cleavage product of motif 1. HBV genotype A has a 2-aa insertion to separate motif 2 from motif 1 (151RRDRGRSPR159), and we found it produced three size forms of HBeAg. Transfection experiments in the HepG2 and Huh7 human hepatoma cell lines established the small, middle, and large forms of HBeAg as cleavage products of motifs 1, 2, and 3 (166RRRR169), respectively. In furin- deficient LoVo cells only the small form was produced. The objective of this R21 grant application is to further evaluate furin as the host factor for HBeAg maturation and a potential therapeutic target. Aim 1 will establish the consequence of furin knockout on HBeAg production from HepG2 and Huh7 cells. Parental cells and knockout clones will be transfected with HBV genomes of genotype A or non- A genotypes, or infected with HBV particles. Aim 2 will establish the consequence of furin silencing or PC inhibition on HBeAg production from a liver progenitor cell line and primary human hepatocytes (PHH). shRNAs against furin will be delivered to differentiated HepaRG cells and PHH. Alternatively, PC inhibitor dec-RVKR-cmk will be added to cell culture. The impact on HBeAg production and genome replication will be determined following HBV infection. Considering that p22 can inhibit HBV DNA replication by forming mixed capsids with core protein, we will also examine whether blocked HBeAg maturation has the added benefit of inhibiting HBV DNA replication. Validating the host enzyme for HBeAg formation and secretion should provide a concrete and non-mutable target for a novel antiviral approach against chronic HBV infection, as potent furin/PC inhibitors have been developed.