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Identification of Cellular Interaction Partners of the Influenza Virus Ribonucleoprotein Complex and Polymerase Complex Using Proteomic-Based Approaches
journal contribution
posted on 2007-02-02, 00:00 authored by Daniel Mayer, Kaaweh Molawi, Luis Martínez-Sobrido, Alexander Ghanem, Stefan Thomas, Sacha Baginsky, Jonas Grossmann, Adolfo García-Sastre, Martin SchwemmleCellular factors that associate with the influenza A viral ribonucleoprotein (vRNP) are presumed to
play important roles in the viral life cycle. To date, interaction screens using individual vRNP
components, such as the nucleoprotein or viral polymerase subunits, have revealed few cellular
interaction partners. To improve this situation, we performed comprehensive, proteomics-based screens
to identify cellular factors associated with the native vRNP and viral polymerase complexes.
Reconstituted vRNPs were purified from human cells using Strep-tagged viral nucleoprotein (NP-Strep)
as bait, and co-purified cellular factors were identified by mass spectrometry (MS). In parallel,
reconstituted native influenza A polymerase complexes were isolated using tandem affinity purification
(TAP)-tagged polymerase subunits as bait, and co-purified cellular factors were again identified by
MS. Using these techniques, we identified 41 proteins that co-purified with NP-Strep-enriched vRNPs
and four cellular proteins that co-purified with the viral polymerase complex. Two of the polymerase-associated factors, importin-β3 and PARP-1, represent novel interaction partners. Most cellular proteins
previously shown to interact with either viral NP and/or vRNP were also identified using our method,
demonstrating its sensitivity. Co-immunoprecipitation studies in virus-infected cells using selected novel
interaction partners, including nucleophosmin (NPM), confirmed their association with vRNP. Immunofluorescence analysis further revealed that NPM is recruited to sites of viral transcription and
replication in infected cells. Additionally, overexpression of NPM resulted in increased viral polymerase
activity, indicating its role in viral RNA synthesis. In summary, the proteomics-based approaches used
in this study represent powerful tools to identify novel vRNP-associated cellular factors for further
characterization.Keywords: Influenza virus • TAP purification • Strep purification • cellular interaction partners • polymerase