Our lab studies Hepatitis B virus (HBV) and Hepatitis C virus (HCV). HBV and HCV are major causes of liver disease, including liver cancer, and together they cause nearly two millions of deaths each year world-wide. HBV is a Hepadnavirus with a DNA genome that replicates by reverse transcription using a virally-encoded reverse transcriptase. HCV is a non-vector borne Flavivirus and has a highly variable RNA genome.
Hepatitis B virus. The key enzyme in HBV replication is its reverse transcriptase. The reverse transcriptase is the main target of anti-HBV drugs. These drugs are nucleoside analogs that are incorporated into the viral DNA and block viral replication. Unfortunately HBV readily evolves resistance to them. Therefore, new drug targets that function by different mechanisms are needed to permit combination drug therapy. To identify such targets, we are studying the reverse transcriptase, usually using the related animal virus, Duck Hepatitis B virus (DHBV), as a model.
We focus on the mechanism by which the reverse transcriptase binds to the viral RNA that is the template for viral DNA synthesis. We identified two motifs on the enzyme that directly bind to the RNA. These data have led us to propose that the reverse transcriptase binds to the viral RNA in a complex, two-stage mechanism. The first stage of RNA binding is promiscuous, with little preference for the viral RNA. Specificity for the viral RNA is proposed to be due to a conformational change in the enzyme that can be induced by the viral RNA but not by other nucleic acids. We are extending these studies at a molecular level, with the hope that learning how to disrupt these RNA binding interfaces will lead to a new anti-HBV drug. Efforts to develop a molecular screen for novel drugs that block RNA binding by the reverse transcriptase are underway.
Hepatitis C virus. HCV is very genetically variable, with six major genotypes and multiple subtypes. The genetic distance between two HCV isolates of the same subtype is larger than the difference between a typical mouse and human gene. We seek to understand how this viral genetic variation affects viral pathology and the response of HCV to antiviral therapy.
HCV is treated with interferon alpha plus ribavirin, but this demanding therapy fails in about half of genotype 1 patients (the major genotype in the USA). We sequenced the HCV genome from 94 patients before therapy, stratified by response to the drugs. Viral genetic variability in patients where the virus was efficiently suppressed was much higher than in patients where suppression was minimal. This implies that the viruses in the patients who responded well were cleared due to the presence of many different variations that independently reduced their ability to counteract the strong interferon response induced by therapy, and that HCV in the poor responders survived because there are only a few ways to optimize activity of the viral proteins (and hence little genetic diversity). We are testing this hypothesis by measuring the interferon-suppressive activities of the variant HCV gene. We are also exploring the possibility that novel genome-wide genetic variation patterns we recently identified may provide a reliable clinical test to predict who will respond to interferon-based therapy.
HCV causes life-threatening illnesses such as cirrhosis or liver cancer in only about 25% of chronically-infected patients, but the reasons why some patients become seriously ill while disease in others is relatively mild are not understood. We hypothesized that this may be due to the presence of HCV genetic variants with varying degrees of virulence. We are conducting two studies to test this hypothesis. First, we are sequencing 120 HCV genomes from 60 patients who had either rapid or slow progression of disease over a 3.5 year period. In the second study we are trying to determine if there are oncogenic variants of HCV. Here, we are sequencing the HCV genomes from 50 patients with or without liver cancer. In both cases the viral genetic analyses will be coupled to functional assays of the variant viral genes to provide biochemical evidence for or against virulence differences among the variants.
Figure 1. Select HCV genes in sequences from patients who responded well to therapy (marked) are more diverse than from the patients who responded poorly to therapy. Importantly, each of these genes can counteract the function of interferon alpha in vitro.