Grant: Project Grant

For viruses to be infectious, they need to be able to pass their genetic material into the cell so that more viruses can be made. This is not a simple task for picornaviruses like poliovirus, because the RNA genome is heavily charged and needs to pass through a hydrophobic lipid membrane. We think this process, known as uncoating, involves a pore that is encoded by the virus, and we plan to examine its structure and functional properties, and get snapshots of it in action. In the same way, if a cell does get infected and start producing viruses, the viral RNA needs to get appropriately packaged into a protein shell. This protein shell, known as a capsid, serves to protect the virus until it is ready to infect another cell. But getting the RNA inside the capsid is no simple task, since only viral RNA should be packaged, and no cellular RNAs. This process, known as encapsidation, takes place deep within the cell and is dependent on a viral protein called 2C. We hypothesize that 2C binds directly to virus capsid precursors and helps package their genomes as they form capsids. Using cryo-electron microscopy, we will determine the structure of poliovirus 2C to see how it can specifically package viral genomes. However, the process of encapsidation as it happens in the cell is difficult to assess structurally. We will use new technology to do just that. By combining a focused ion beam with cryo-electron tomography - a first in Canada - we can see molecular-level interactions in 3-dimensions in the center of a frozen cell. Using this approach, we will examine "virus factories" in the cell, and see how 2C interacts with the surrounding membranes. RNA entering the virus during encapsidation, and RNA exiting the virus during infection, are processes that act as bookends of the viral life cycle. By understanding the molecular mechanisms involved, we hope to find ways of inhibiting these processes essential to picornaviruses, and other related viruses.