July 15 | 2013

Exploration of binary virus-host interactions using an infectious protein complementation assay

Authors | Sandie Munier, Thomas Rolland, Cédric Diot, Yves Jacob, and Nadia Naffakh

Abstract | A precise mapping of pathogen-host interactions is essential to achieve a comprehensive understanding of the processes of infection and pathogenesis. The most frequently used techniques for interactomics are the yeast two-hybrid binary methodologies, which does not recapitulate the pathogen life cycle, and the TAP-MS co-complex methodologies, which cannot distinguish whether interactions are direct or indirect. New technologies are thus needed to improve the mapping of pathogen-host interactions. Here, we detected binary interactions between influenza A virus polymerase and host proteins during the course of an actual viral infection, using a new strategy based on trans-complementation of the Gluc1 and Gluc2 fragments of Gaussia princeps luciferase.

Infectious recombinant influenza viruses that encode a Gluc1-tagged polymerase subunit were engineered to infect cultured cells transiently expressing a selected set of Gluc2-tagged cellular proteins involved in nucleocytoplasmic trafficking pathways. A random set and a literature-curated set of Gluc2-tagged cellular proteins were tested in parallel. Our assay allowed sensitive and accurate recovery of previously described interactions while it revealed 30% of positive, novel viral-host protein-protein interactions within the exploratory set. In addition to cellular proteins involved in nuclear import pathway, components of the nuclear pore complex such as NUP62 and mRNA export factors such as NXF1, RMB15B and DDX19B were identified for the first time as interactors of the viral polymerase.

Gene silencing experiments further showed that NUP62 is required for efficient viral replication. Our findings give new insights on the subversion of the host nucleocytoplasmic trafficking pathways by influenza A viruses. They also demonstrate the potential of our infectious protein complementation assay for high-throughput exploration of influenza virus interactomics in infected cells. With more infectious reverse genetics system becoming available, this strategy should be widely applicable to numerous pathogens.

Mol Cell Proteomics  Papers in Press. Published on July 1, 2013 as Manuscript M113.028688.