MODEL SYSTEMS FOR ANALYSIS OF HBV REPLICATION

Hepatitis B virus (HBV) represents a serious worldwide health problem. The
acute infection is usually self-limiting but may prove fatal, and, in
addition, up to 10% of infected individuals become chronic carriers of the
disease. The total number of carriers has been estimated at 280 million.
Chronicity frequently progresses to cirrhosis the liver and hepatocellular
carcinoma. Chronic carriers represent the main reservoir of the virus, with
transmission occurring through contaminated blood products, IV drug abuse,
sexual contact, and from mother to infant at birth. A precise understanding
of the mechanism of viral replication is required to devise strategies for
eliminat-ing the carrier state. This proposal will address several
unresolved issues pertaining to the replication of the viral genome and
viral morphogenesis. The first specific aim is to examine core protein
mutants for assembly and for encapsidation of pregenomic RNA, polymerase,
and polymerase-RNA complexes. The proteins will be expressed in insect
cells using a baculovirus expression system or in in vitro translation
systems and in vitro assays will be developed to examine these functions.
The ability of core mutants to complement the replication of a core minus
HBV mutant will be examined in the human hepatocellular carcinoma cell
line, HepG2. The mechanism of encapsidation and the functional significance
of the virion associated kinase will be explored. The second specific aim
will be to express polymerase and polymerase functional domains in insect
cells and to develop in vitro assays for reverse transcription, pregenomic
RNA binding, nucleotide binding, and RNase H activity. The third specific
aim will be to examine the RNA sequence requirement for encapsidation of
pregenomic RNA by core and for interaction of pregenomic RNA with
polymerase. The in vitro RNA transcripts used in assays for core and
polymerase functions will be altered to determine the minimal sequences
required for recognition. The final specific aim will be to utilize the
information obtained in the in vitro assays to make specific mutations in
the HBV genome and examine replication the mutants in HepG2 cells.