The goal of this project is to understand the molecular mechanisms by which a virus, vesicular stomatitis virus (VSV) replicates. VSV has a similar RNA genome to other potent human and animal pathogens such as Ebola and measles. When VSV enters a host cell it synthesizes an enzyme, RNA dependent RNA Polymerases (RdRPs) that copies the viral RNA genome. This project will examine the mechanism by which RdRPs read and copy the viral genome template in infected cells, which is critical for the replication of viruses. The results from this study will improve scientific understanding of how RdRPs work and identify potential target mechanisms for inhibition of viral infections. This study will train students who are traditionally underrepresented in STEM fields, by engaging them within teams doing research at the University of Utah and University of Massachusetts Medical School at Worcester. The proposed activities will also support development of Biophysics courses that will benefit undergraduate interdisciplinary education, and encourage participation of K-12 students in science by communicating aspects of the scientific results through creation of a "virus dance" project.
This project will use single molecule live cell imaging to visualize transcription events from single VSV genome templates in infected cells and utilize fluorescence correlation spectroscopy to measure the concentration of free RdRPs during transcription in vivo. When stretched, the genome template of VSV is more than 4 microns in size and RdRPs can only initiate transcription on its 3' end. While 50 RdRPs are tightly bound to this template, how they initiate and sustain transcription is not clear. The live cell imaging experiments will determine the number of active RdRPs per genome and their cooperativity. Measurements of the concentration of free RdRPs not bound to the genome will verify if a dissociation and 3' binding mechanism can support viral transcription in vivo. This project will also use reconstituted templates in vitro to visualize the mechanism by which RdRPs redistribute on the genome template. We have observed single RdRP sliding on purified genome templates in vitro. In this project we will further characterize the sliding mechanism of RdRPs on genome templates. By measuring the dynamics of RdRPs in vivo as well as in vitro this project will determine the mechanism of transcription by RdRPs during VSV RNA virus infection.
The project was funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.
