Study of novel pro-viral activities of non-structural protein 1 (NS1) of influenza A virus

Abstract

La proteína NS1 del virus de la gripe A desempeña múltiples funciones províricas en células infectadas. Esta proteína multifuncional interactúa con numerosos factores celulares, algunos de relevancia biológica aún poco definida, como su capacidad para unirse y activar las PI3K de clase IA. Las PI3K son quinasas lipídicas reguladas que actúan como nodos clave en redes de señalización celular. La activación de PI3K por NS1 se produce mediante su unión a la subunidad p85β, aunque la importancia de esta especificidad y la contribución de las isoformas de p110 siguen siendo desconocidas. Para estudiarlo, hemos desarrollado un ensayo BiFC que permite seguir heterodímeros específicos de PI3K. Nuestros resultados muestran que NS1 induce una relocalización y activación dependientes de la isoforma de los heterodímeros de PI3K. Además, mutaciones oncogénicas en ambas subunidades imitan el efecto de NS1 y rescatan parcialmente la pérdida de función en virus mutantes

The non-structural protein 1 (NS1) of influenza A virus performs a broad variety of proviral activities in the infected cell, primarily mediating evasion from the host innate immune response by being the main viral interferon antagonist. However, among the multiple interactions described for this small multifunctional protein, there are several whose biological relevance remains obscure, such as the ability of NS1 to bind and activate class IA phosphoinositide 3kinases (PI3Ks). PI3Ks are highly regulated lipid kinases that act as critical nodes in multiple cell signaling networks and regulate cellular physiology, including differentiation, growth, survival, trafficking, and immune function. As such, PI3Ks are also important proto-oncogenes whose deregulation lies behind a substantial number of different human cancers. Structurally, class IA PI3Ks are heterodimers formed by a regulatory (p85) and catalytic (p110) subunit, of which there are several isoforms described, adding further layers of complexity to their activity. Activation of PI3K by NS1 is mediated by NS1 binding to the p85 subunit. Interestingly, such binding is specific to the p85β isoform, and PI3K heterodimers containing p85β and any of the other three p110 isoforms are presumably activated by NS1. However, the significance of this p85β specificity, and the contributions of different p110 isoforms, remains unknown. In order to better understand the consequences of PI3K activation by NS1, we have developed a bimolecular fluorescence complementation (BiFC) assay to selectively track the different PI3K heterodimers according to their specific regulatory and catalytic isoforms, as well as to assess their behavior upon activation. Using this system, we found that NS1 induces an isoform-specific relocation and activation of the different PI3K heterodimers. However, the effects of other known activators of PI3K, such as Ras and Src, were different from those induced by NS1. We found that clinically relevant, oncogenic hyper-activating mutations in both catalytic and regulatory subunits of PI3K could mimic the effect caused by NS1, and partially rescued the loss of viral fitness in a recombinant virus encoding a p85β-binding deficient NS1. We postulate that by mimicking an oncogenic deregulation of the PI3K pathway, influenza A virus induces a transient, transformed-like status in the infected cell to stimulate virus replication