MECHANISMS OF HEPADNAVIRUS ASSEMBLY AND REPLICATION

Project: Research project

Project Details

Description

Hepadnaviruses, or hepatitis B viruses, are small, hepatotropic DNA
viruses that replicate through an RNA intermediate (the pregenomic RNA
or pgRNA) by reverse transcription. A critical early step in viral
replication is the assembly of a ribonucleoprotein (RNP) complex
comprised of the viral reverse transcriptase (RT) and pgRNA, which
triggers both the assembly of nucleocapsids, the site of viral DNA
synthesis, and the initiation of reverse transcription, which is primed
by RT itself (the protein priming reaction). Through a complex multi-
step reverse transcription pathway, the pgRNA is subsequently converted,
within the context of the nucleocapsids, into the characteristic,
relaxed circular DNA genome which can then exit the cell. The broad,
long term objective of this proposal is to elucidate the molecular
mechanisms of hepadnavirus replication, focusing on the virus-cell
interactions critical for viral assembly and replication. The first
specific aim is to identify host factors required for the interaction
between RT and pgRNA and for protein priming. A combined approach based
on physical association with RT, pgRNA and viral particles and
biochemical fractionation and reconstitution will be used for this
purpose, taking advantage of an established cell-free system that can
recapitulate these early events in viral replication. The second aim
is to elucidate the determinants on both RT and pgRNA responsible for
specific RNA-protein interaction. Optimal conditions for RT- binding
and crosslinking will be sought. The RT and contact sites in the RNP
complex will then be identified through protein and RNA sequence
analyses. This will, in turn, guide further mutagenesis studies to
elucidate the specific requirements of RT and pgRNA for RNP formation.
The third aim is to determine the requirements for individual steps of
the viral reverse transcription pathway, using a recently developed
synchronized viral replication system. Nucleocapsids from every stage
of reverse transcription will be isolated to determine their structural
changes, which accompany, and may be required for, the progression of
viral DNA synthesis. Furthermore, specific host factors required for
the different steps of reverse transcription will be identified by (1)
carrying out viral DNA synthesis with the isolated nucleocapsids under
cell-free reconstitution conditions and (2) treating virus-producing
cells, at different stages of reverse transcription, with agents that
target candidate host factors. The accomplishment of these goals should
reveal novel cellular, as well as viral, targets for developing
effective antivirals which is urgently needed for the treatment of over
300 million patients worldwide infected with the hepatitis B virus, who
are at great risk of developing hepatic failure and hepatocellular
carcinoma. In addition, studies on how host factors facilitate viral
replication will also provide important insights into the normal
cellular functions of these factors.
StatusActive
Effective start/end date2/15/998/31/21

Funding

  • National Institute of Allergy and Infectious Diseases: $199,373.00

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