We are interested in establishing the impact of hepatitis B virus genotypes and naturally occurring mutations on viral genome replication, protein expression (core protein and e antigen), and virus particle release. It is well documented that certain mutations and some viral genotypes are closely associated with the development of liver cancer, but the molecular mechanisms remain unknown. We use molecular approaches to address this important medical problem.

Hepatitis B virus (HBV): chronically infects nearly 400 million people worldwide, leading to viral hepatitis, cirrhosis, and liver cancer. HBV is endemic in Asia, where the virus usually transmits from HBeAg+ mother to infants. HBV related liver cancer is a leading cause of death among males in this part of the world. Asian patients are infected primarily by genotypes B and C. Over the course of decades of infection, the wild-type virus is gradually replaced by various mutants, including precore mutants and core promoter mutants. HBV isolated from most liver cancer patients harbor core promoter mutations.

Experimental approaches: cloning of full-length HBV genomes from patient sera or making chimeric constructs or site-directed mutants; generation of replication competent form (dimer); transfection into human hepatoma cells; analysis of protein expression, genome replication, and virion secretion.

Research projects:

1. Regulation of HBeAg expression by naturally occurring mutations. Known and potential mutations include those affecting transcription of HBeAg mRNA, translation initiation, premature chain termination, posttranslational cleavage (signal peptide removal and carboxyl terminus removal), maintenance of HBe antigenicity by a disulfide bond. Current interest is to identify the enzyme responsible for carboxyl terminal cleavage.

2. Regulation of genome replication. We are interested in determining whether the common precore mutation that abolishes HBeAg expression enhances genome replication. We would like to establish the molecular mechanisms whereby core promoter mutations enhance genome replication. We hypothesize that enhanced genome replication brought about by the core promoter and/or precore mutations elevate the risk of developing liver cancer.

3. Regulation of virion secretion. We have identified amino acid substitutions in the core protein and envelope proteins that impair virion secretion. We have also identified an amino acid substitution in the envelope proteins that can overcome the secretion defect induced by other mutations in the same proteins. In addition, we found certain point mutations in the core protein can affect the genome maturity of secreted virions. Future studies aim to elucidate the mechanisms involved.

4. Characterization of virion secretion property of the immune escape mutants. Current HBV vaccine is made of the major envelope protein and induces neutralizing antibodies. The vaccine fails in some people due to the emergence of the so-called "immune escape mutants" containing amino acid substitutions in the neutralizing epitopes. We hypothesize that immune escape mutants will not overtake the wild-type virus because they are compromised in virion secretion. Experiments are being conducted to test this hypothesis.

Current Support:

American Cancer Society (ACS) Research Scholar Award: #RSG 06-059-01-MBC
1/06 – 12/09
direct costs: 150,000 USD/year
"Hepatitis B virus replication and secretion."
Goal of the project: to determine how the core promoter mutations enhance HBV genome replication while inhibiting HBeAg expression; to investigate the mechanisms whereby mutations in the S gene regulate virion secretion; to evaluate the pathogenicity of high genome replication and/or defective virion / HBsAg secretion to the hepatocytes.
Role in the project: Principal Investigator

National Institutes of Health (NIH) / National Cancer Institute (NCI): R01 CA109733
4/05-3/10
direct costs: 153,000 USD/year
"Molecular targets for interruption of HBV Infection"
The major goal of this project is to evaluate duck hepatitis B virus receptor (carboxypeptidase D) and co-receptor (glycine decarboxylase) as potential targets for interruption of DHBV infection at the early stage of the viral life cycle.
Role in the project: Co-investigator

Completed Support:

NIH/ National Center for Research Resources (NCRR): P20RR15578
9/01-8/05
"Center of Biomedical Research Excellence" (PI: John Sedivy).
Goal of the project: this project funds core facilities and research projects at Brown University and its affiliated hospitals.
Role in the project: Co-investigator.

NIH/ National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK): R21DK62857
(200,000 USD)
7/03-4/05
"Replication and secretion of hepatitis B virus variants."
Goal of the project: to identify naturally occurring mutations that modify viral genome replication and virion secretion. Results obtained from this project constitute the preliminary results of the current application.
Role in the project: Principal Investigator.

NIH/NIDDK: R03AI54535
(100,000 USD)
4/03 –3/05
"Hepatitis B virus e antigen expression." 4/03-3/05.
Goal of the project: to search for novel mutations that regulate HBeAg expression.
Role in the project: Principal Investigator.

Lifespan Developmental Award
(45,000 USD)
4/02-9/03
"Molecular basis for unusual replication and secretion phenotypes of hepatitis B virus variants."
Goal of the project: to initiate investigations on why some core promoter mutants of HBV display marked genome replication and impaired virion secretion.
Role in the project: Principal Investigator.

Guarnieri M., Kim K., Bang G., Li J., Zhou Y., Tang X., Wands J., Tong S. 2006. Point mutations upstream of hepatitis B virus core gene affect DNA replication at the step of core protein expression. J. Virol. 80: 587-595.

Bang G., Kim K., Guarnieri M., Zoulim F., Kawai S., Li J., Wands J., Tong S. 2005. Effect of mutating the two cysteines required for HBe antigenicity on hepatitis B virus DNA replication and virion secretion. Virology 332: 216-224.

Khan N., Guarnieri M., Ahn S., Li J., Zhou Y., Bang G., Kim K., Wands J., Tong S. 2004. Modulation of hepatitis B virus secretion by naturally occurring mutations in the S gene. J. Virol. 78: 3262-3270.

Ahn S.H., Kramvis A., Kawai S., Spangenberg H., Li J.S., Kimbi G., Kew M., Wands J., Tong, S. 2003. Sequence variation upstream of precore translation initiation codon reduces hepatitis B virus e antigen production. Gastroenterology 125: 1370-1378.

Parekh S., Zoulim F., Ahn S.H., Tsai A., Li J.S., Kawai S., Khan N., Trepo C., Wands J.R., Tong, S. 2003. Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promotermutants. J. Virol. 77:6601-6612.

Spangenberg H.C., Lee H.B., Li J.S., Tan F., Skidgel R., Wands J.R., Tong S. 2001. A short sequence within domain C of duck carboxypeptidase D is critical for duck hepatitis B virus binding and determines host specificity. J. Virol. 75:10630-10642.

1989: Young Investigator's Award from the French Society for the Study of the Liver (AFEF).
2004: Session Chair: Variants session, International Annual Molecular Biology of Hepatitis B Virus Meeting, Woods Hole, Mass. (October).
2005: Ad Hoc member: National Institutes of Health (NIH) Center for Scientific Review Special Emphasis Panel on Innovative Virology (March 17-18).
2006: Guest Speaker. Satellite Symposium on Hepatitis B Virus Genotypes / 42nd Annual Meeting of Japanese Society of Hepatology, Kyoto, Japan (May 25-28).
2006: Session Chair: HBV mutants. 12th International Symposium on Viral Hepatitis and Liver Diseases. Paris, France (July 1-5).

Member, American Society for Microbiology
Member, American Society for the Study of Liver Diseases