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Principal Investigator: SHUPING TONG
Organization: RHODE ISLAND HOSPITAL
Fiscal Year: 2021
Award: $245,875
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <AAV vector><Abscission><Amino Acids><Antiviral Agents><Antiviral Drugs><Antiviral Therapy><Antivirals><Applications Grants><Arginine><Burkitt Herpesvirus><Burkitt Lymphoma Virus><C-terminal><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Capsid><Cas nuclease technology><Cell Body><Cell Culture Techniques><Cell Line><CellLine><Cells><Chickens><Chronic><Chronic Hepatitis B><Cleaved cell><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Codon><Codon Nucleotides><Core Particle><Core Protein><DNA Replication><DNA Synthesis><DNA biosynthesis><Development><E-B Virus><EB virus><EBV><Enzyme Gene><Enzymes><Epstein Barr Virus><Excision><Extirpation><Gallus domesticus><Gallus gallus><Gallus gallus domesticus><Genes><Genome><Genotype><Goals><Grant Proposals><HBV><HBV Genotype><HBcAg><HBeAg><HHV-4><HHV4><Hep G2><HepG2><HepG2 cell line><Hepatic Cancer><Hepatic Cells><Hepatic Parenchymal Cell><Hepatitis B><Hepatitis B Core Antigen><Hepatitis B Infection><Hepatitis B Virus><Hepatitis B e Antigens><Hepatitis Be Antigens><Hepatocarcinoma><Hepatocellular Carcinoma><Hepatocellular cancer><Hepatocyte><Hepatoma><Homologous Serum Hepatitis Virus><Host Factor><Host Factor Protein><Human><Human Herpesvirus 4><Immune Tolerance><Immune response><Immunologic Tolerance><Immunological response><In Vitro><Infectious Mononucleosis Virus><Integration Host Factors><Knock-out><Knockout><L-Arginine><Lentiviral Vector><Lentivirus Vector><Liver><Liver Cells><Liver Cells Carcinoma><Malignant neoplasm of liver><Modern Man><Mutation Analysis><N-terminal><NH2-terminal><Nucleosome Core><Nucleosome Core Particle><Nucleotides><Pathway interactions><Peptide Signal Sequences><Primary carcinoma of the liver cells><Production><Progenitor Cells><Proprotein Convertases><Proteins><Removal><Risk><Series><Signal Peptide><Signal Sequences><Site><Strains Cell Lines><Structure><Surgical Removal><Testing><Therapeutic><Therapeutic Intervention><Transfection><Translating><Translation Initiation><Viral Diseases><Viral Genome><Viral Hepatitis B><Viral Receptor><Virion><Virus Diseases><Virus Particle><Virus Receptors><Virus Replication><adeno-associated viral vector><adeno-associated virus vector><aminoacid><anti-hepatitis B><anti-viral agents><anti-viral drugs><anti-viral therapy><anti-virals><base><cell culture><chronic HBV infection><chronic hepatitis B virus infection><chronic infection><cleaved><cultured cell line><developmental><e Antigens><established cell line><experiment><experimental research><experimental study><hepatic body system><hepatic organ system><hepatitis B virus genotype><hepatoma cell><host response><immune system response><immune system tolerance><immune unresponsiveness><immunogenic><immunological paralysis><immunoresponse><in vivo><infection with HBV><infection with hepatitis B virus><inhibitor><inhibitor/antagonist><intervention therapy><knockout gene><liver cancer><liver carcinoma><malignant liver tumor><novel><pathway><persistent infection><prevent><preventing><protein signal sequence><reconstitute><reconstitution><resection><response><seroconversion><serum hepatitis><shRNA><short hairpin RNA><small hairpin RNA><stem cells><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><therapeutic target><trans-Golgi Network><viral infection><viral infectious disease treatment><viral multiplication><viral replication><virus genome><virus infection><virus multiplication><virus-induced disease>