Identification of Cellular Interaction Partners of the Influenza Virus Ribonucleoprotein Complex and Polymerase Complex Using Proteomic-Based Approaches

Abstract

SummaryCellular factors which 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 (NPStrep) 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 which co-purified with NP-Strepenriched vRNPs, and 4 cellular proteins which 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 virusinfected 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 vRNPassociated cellular factors for further characterization.

Influenza A Virus Multiplication and the Cellular SUMOylation System

“…Four different viral-host interaction screenings [89,93,95,97] identified subunits of the cellular RNA Polymerase II (Pol II), which further reinforced previous work performed on the proposed mechanisms for the role of Pol II on viral transcription. During the normal cycle of cellular RNA transcription, the promoter associated Pol II is phosphorylated by TFIIH to initiate transcription.…”

“…Moreover, in vitro analysis of viral transcription revealed about a ~5-fold reduction in the fraction of viral polyadenylated (positive sense) transcripts, suggesting that SFPQ/PSF seems to facilitate the polyadenylation of viral transcripts [113]. UAP56, (Bat1/Raf-2p48), a known viral-interactor protein that was also identified in a vRdRp interaction screening by Mayer et al [89], is a fairly well established RNA helicase involved in spliceosome assembly [114], facilitating the nuclear export of cellular mRNA [115], and is a constituent of the transcription export complex which delivers pre-mRNAs bound to the exon-junction complex to nuclear export factor 1 (NXF1) [116]. Although UAP56 is clearly a factor in cellular RNA splicing, in vitro studies indicate that UAP56 forms heterodimers with NP in the absence of vRNA.…”

“…However, up to fairly recent times, most studies related to influenza replication and multiplication focused exclusively on the mechanistic assessment of processes mediated by viral proteins and put little emphasis on the cellular factors or pathways involved in influenza replication. During the last few years, the use of multiple approaches such as protein pull-downs, chemical inhibition studies, yeast-two hybrid screening, affinity purification, and RNA interference (RNAi), have led to the identification of multiple cellular factors essential for influenza viral replication [89][90][91][92][93][94][95][96][97]. In the sections below, we will review the most recent findings related to the relevance of specific host cellular proteins and systems for influenza transcription and replication.…”

Oligomerization of the influenza virus polymerase complex in vivo

“…On the contrary, replication of viral RNPs does not require capped primers and occurs by the synthesis of a complementary RNA (cRNA) intermediate, which is a complete copy of the virion RNA (vRNA) (Hay et al, 1982), and serves as template for the generation of progeny vRNAs. Both vRNAs and cRNAs are encapsidated into RNP structures during replication (reviewed by Elton et al, 2005).In addition to the RNP-associated polymerase, soluble polymerase complexes have been detected in infected cells (Detjen et al, 1987) and the interaction of cellular proteins with the polymerase complex has been described (Deng et al, 2006;Mayer et al, 2007;Momose et al, 2002;Naito et al, 2007) (N. Jorba and others, unpublished data). To characterize these soluble intracellular polymerase complexes we have addressed their expression and purification from human cells.…”

Cell cycle independent role of cyclin D3 in host restriction of influenza virus infection

“…From RNA interference (RNAi)-based genome-wide screening strategies in Drosophila (5) and mammalian systems (6 -9) to other approaches, including proteomics (10,11), yeast two-hybrid (Y2H) 3 analyses (7,12), and microarrays (7,(13)(14)(15), extensive information has been acquired. Multiple cellular networks have been implicated in influenza virus replication through either direct protein-protein interactions (10,16) or signaling pathways (17). Although these strategies unveiled hundreds of genes important for the virus life cycle, their functional relevance and molecular mechanisms are still poorly understood.…”