The research interests of this laboratory focus on understanding human virus-host cell interactions. We use molecular biology, virology, structural biology, and chemical biology approaches to study RNA-protein interactions in dengue fever virus, West Nile virus, and hepatitis C virus (HCV).
How do host cells sense that they have been invaded by a pathogen? Recent evidence suggests that structured viral RNAs can be Pathogen Associated Molecular Patterns (PAMPs) that are recognized by host Pattern Recognition Receptors (PRR) to activate cell signaling. We recently identified a 100-nucleotide polyU/UC domain in the HCV 3’ untranslated region RNA that is a potent activator of innate immune signaling (a PAMP) to interferon expression. This polyU/UC RNA binds to RIG-I, a cytoplasmic signaling molecule (a PRR) that is independent of toll-like receptors. Furthermore, we discovered that polyU/UC RNAs that are modified with 2’-fluorodeoxyuridine or pseudouridine do indeed bind to RIG-I, but they are blocked in signaling downstream to interferon. We are using this unique RNA tool to probe the mechanism of RNA-mediated activation of innate immune signaling during virus infections.
We are collaborating with Sangeeta Bhatia and Daniel Anderson (MIT) to use a new human primary liver culture system to study the immunomodulatory effects of the polyU/UC RNA. We have demonstrated that the primary human hepatocytes can be transfected with polyU/UC RNAs by using a new type of transfection reagent (“lipidoids”), and that the hepatocytes respond by expressing interferon. Ongoing experiments will challenge the cells receiving the polyU/UC RNAs with HCV and dengue fever viruses to determine if the cells are protected from infection by the up-regulated innate immune state.
There is currently no commercial vaccine for HCV or dengue fever virus infections, and it is likely that a variety of drugs will be useful in fighting these complex diseases. We are using high throughput screening to identify small molecules that block viral RNA translation or replication. To date, over 8000 small molecules have been screened, and six chemotypes of inhibitors have been identified. Current work is focused on secondary screening and identifying the mechanism of action. The small molecules will also be tested in the human primary liver culture system described above.
Non-polyadenylated viral RNAs infect both animals and plants, and we seek to understand common cross-kingdom features of the translational apparatus that support expression of these structurally distinct templates. Although the mRNAs lack a poly(A) tail, biochemical and functional evidence suggests that poly(A) binding protein (PABP) is functional in translating both poly(A)+ and poly(A)- mRNAs. We are testing a hypothesis stating that PABP binds to non-polyA nucleotide sequences in the 3’ untranslated region, allowing them to compete with cellular polyA RNAs for the translational machinery. When PABP is depleted by siRNA treatment, viral protein accumulation is diminished without a significant effect on host cell translation. These data suggest that low affinity PABP binding to non-adenylated RNAs is important for translation, and that the absence of the polyA tail is significant for accurate RNA replication.
1. Uzri, D., and L. Gehrke. 2009. Nucleotide sequences and modifications that determine RIG-I/RNA binding and signaling activities. J Virol 83:4174-4184.
2. Gomila, R. C., G. W. Martin, and L. Gehrke. 2011. NF90 binds the dengue virus RNA 3' terminus and is a positive regulator of dengue virus replication. PLoS One 6:e16687.
3. Schmidt, D. J., B. E. Pickett, D. Camacho, G. Comach, K. Xhaja, N. J. Lennon, K. Rizzolo, N. de Bosch, A. Becerra, M. L. Nogueira, A. Mondini, E. V. da Silva, P. F. Vasconcelos, J. L. Munoz-Jordan, G. A. Santiago, R. Ocazionez, L. Gehrke, E. J. Lefkowitz, B. W. Birren, M. R. Henn, and I. Bosch. 2011. A phylogenetic analysis using full-length viral genomes of South American dengue serotype 3 in consecutive Venezuelan outbreaks reveals a novel NS5 mutation. Infect Genet Evol 11:2011-2019.