Eric Jones with Fred Porter and Dr. Ann Palmenberg

Institute for Molecular Virology, UW-Madison

Nucleocytoplasmic c transport, the process of transporting macro molecules in and out of the nucleus within eukaryotic cells is carried out across the nuclear membrane by Nuclear Pore Complexes (NPCs) and a collection of other proteins. This process is responsible for ensuring that a cell is capable of carrying messages (ex. proteins and DNA) between the nucleus and the cytoplasm so the cell can function normally.  This process is carried RAN, which is an energy dependent enzyme that uses GTP as an energy source to induce active transport for information in and out of the nucleus.   Cardioviruses such as Encephalomyocarditis Virus (EMCV) encode a unique protein know as the Leader that inhibits the traffic of macromolecules in and out of the nucleus by binding RAN and interfering with its activity.  By doing this, EMCV has created an effective way of preventing the cell from mounting a specific defense against the virus. 

There are several phosphorylation sites on the Leader protein which have been located.   We hypothesized that one particular phosphorylation site, threonine 47, is important for the inhibition of nucleocytoplasmic transfer.   To test this, we compared wild-type Leader protein to T47 Alanine mutant in a series of tests which included RAN binding pull-downs and a phosphorescent nuclear import assay.  The T47A mutant shows no difference in RAN binding, but shows a reduction in its ability to inhibit nuclear transfer.  This suggests that Leader may have multiple ways of inhibiting nucleocytoplasmic transfer and preventing the cell from fighting the infection.