Our lab studies the Hepatitis B virus (HBV) and Hepatitis C virus (HCV). HBV and HCV are major agents of liver disease and liver cancer, causing millions of deaths each year world-wide. Despite the similarity in their names, HBV and HCV are very different viruses. HBV is a small virus with a DNA genome that replicates by reverse transcription using a virally-encoded reverse transcriptase. HCV is a larger virus with an RNA genome that is replicated by a virally-encoded RNA-dependent RNA polymerase. Despite these differences, HBV and HCV cause very similar diseases in people.
Hepatitis B virus.
We are pursuing two lines of research into the role of the reverse transcriptase
in HBV replication using the closely related animal virus, Duck Hepatitis
B virus (DHBV), as a model.
The first line of research is a structure-function analysis of the reverse transcriptase (P), employing functional recombinant enzyme. We discovered that P initiates reverse transcription at a previously unknown location on the viral genome and employs a novel strand transfer reaction during first strand DNA synthesis. We also discovered that the RNA stem-loop that forms the viral origin of DNA synthesis has a unique function, in that it induces a conformational change in P that is needed for it to mature to an enzymatically active form. We are extending this research by determining the nature of the alteration to the structure of P and by using a variety of approaches to determine which motifs of P contribute to its various biochemical activities.
Figure Legend. Metabolism of the DHBV polymerase. The polymerase is translated and then either binds to an RNA motif on the viral RNA (epsilon), undergoes enzymatic activation through a conformation alteration, and is then encapsidated into nascent core particles (right arm of figure), or it binds to cytoplasmic components and is post translationally modified (left arm of figure). The number of polymerase molecules entering the left arm vastly exceeds the number entering the right arm.
The second line of research is a characterization of the distribution and structure of P in viruses and in cells. We have made three observations that drastically change the way we view P. First, we discovered that contrary to all expectations, P accumulates to easily detectable levels outside of viral particles. This non-encapsidated P is bound to a cytoplasmic structure in multiple complexes. Our second observation rests on kinetic analyses of the synthesis and degradation of P and the core protein (the protein that makes the particle in which reverse transcription occurs). These studies revealed that the vast majority of P molecules are never encapsidated and that P is translated unexpectedly rapidly from a suboptimal postion on a bicistronic mRNA. Our third observation is that the intracellular accumulation of P is regulated post-translationally. Together, these observations indicate that P has at least two metabolic fates, to be encapsidated or to remain non-encapsidated (see Figure). Although it is essential for viral replication, the encapsidation pathway accounts for only a trivial proportion of the total number of P molecules synthesized. These results raise the possibility that P may have additional role(s) in hepadnaviral replication beyond its well-known role in copying the viral genome, and this possibility is under investigation.
Hepatitis C virus.
HCV infections are treated with a combination of interferon alpha and ribavirin.
This therapy is effective in approximately 40% of white patients, but it appears
to work in less than 20% of African Americans. Our lab recently entered the
HCV field by joining the Virahep-C multi-center research project sponsored
by the NIDDK aimed at determining the reasons for the poor response of HCV
to antiviral therapy in African Americans. We will perform whole-genome sequencing
of HCV to assess the variability in the genomes. The HCV genome is known to
be highly variable, and these variations are believed to underlie much of
HCV's biology. These genomic sequences will be analyzed for clues to the reasons
for the poor response rate in African Americans. We will also express recombinant
RNA-dependent RNA polymerases from representative variants and assess their
function biochemically. We will do this is because the variability of the
HCV genome is a direct product of the polymerase's activity, and hence any
change in the activity of the RNA polymerase could directly result in alterations
to the spectrum of HCV variants found in the patients. These studies are just
getting underway.