The following application will focus on determining the mechanism by which the influenza A protein, NS1, blocks nuclear RNA-export function. This Inhibition of cellular mRNA export is an integral component of the influenza A offensive repertoire against its cellular host. In doing so, the virus prevents the host cell from building its usual innate defense machinery. Export of most cellular mRNAs is mediated by the transport receptor Tap. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporins. Over expression of Tap can counteract the NS1 RNA export block. We have new evidence that NS1 can bind Tap directly and propose that the inhibitory effects of NS1 on RNA transport requires direct interaction between NS1 and Tap.
Our first aim will be to directly test this hypothesis by generating a NS1 mutant deficient for Tap binding. This mutant can then be used to differentiate between direct and indirect effects of NS1 on the Tap export pathway. The following application also plans on examining the exact mechanism by which NS1 interferes with Tap function.
The second aim will be to test the possibility that NS1 causes a decreased in Tap protein levels or mis-localization of Tap away from its site of action. We will also determine the exact site within cells where Tap and NS1 interact using novel bimolecular fluorescence complementation assay to help reveal the specific steps within Tap export pathway inhibited by NS1. We will also examine the possibility that NS1 prevents the heterodimerization of Tap with its co-factor, Nxt1 or its self-oligomerization in vivo and in vitro. The formation of both Tap/Nxt1 and Tap/Tap complexes are required for efficient RNA export. We will also examine the ability of Tap to interact with the nuclear pore complex in the presence of NS1.
Our third aim will focus on the possible interference of NS1 on the recruitment of RNA cargo to Tap. Understanding the exact mechanism of NS1-mediated RNA export would not only allow us to predict the most virulent IAV strain but would also give us information that could lead to the development of novel anti-viral compounds.
The influenza A protein NS1 disarmed infected cells by preventing the export of cellular mRNAs that encode anti-viral cellular factors. Most cellular mRNAs are exported out of the nucleus through the Tap pathway. This application will examine the exact mechanism(s) by which NS1 interferes with Tap-mediated RNA export.