Abstract
A precise mapping of pathogen-host interactions is essential for comprehensive understanding of the processes of infection and pathogenesis. The most frequently used techniques for interactomics are the yeast two-hybrid binary methodologies, which do not recapitulate the pathogen life cycle, and the tandem affinity purification mass spectrometry co-complex methodologies, which cannot distinguish direct from indirect interactions. New technologies are thus needed to improve the mapping of pathogen-host interactions. In the current study, 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 the sensitive and accurate recovery of previously described interactions, and it revealed 30% of positive, novel viral-host protein-protein interactions within the exploratory set. In addition to cellular proteins involved in the 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 regarding the subversion of 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 systems becoming available, this strategy should be widely applicable to numerous pathogens.
Highlights
From the ‡Institut Pasteur, Unitede Genetique Moleculaire des Virus a ARN, Departement de Virologie, F-75015 Paris, France; §CNRS, UMR3569, F-75015 Paris, France; ¶Universite Paris Diderot, Sorbonne Paris Cite, Unitede Genetique Moleculaire des Virus a ARN, EA302, F-75015 Paris, France; ʈCenter for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215; **Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115
We identified karyopherin alpha 5 (KPNA5), Ran GTPase activating protein 1 (RANGAP1), NUP62, and NUP62CL as additional partners
KPNA5 shares more than 90% similarity with KPNA1 and KPNA6, and human KPNA5 expression seems to be restricted to the testis [43]
Summary
Cells and Viruses—293T, A549, and BSR cells were grown in complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS). For subsequent amplification of the recombinant PB1-, PB2-, and PA-Gluc or PB1-, PB2-, and PA-Gluc viruses, MDCK cells were infected at an MOI of 0.0001 and incubated for 3 days at 35 °C in DMEM containing TPCK-treated trypsin at a concentration of 1 g/ml. At 17 h post-transfection, cells were rinsed with DMEM and infected with 50 l of a viral suspension containing 9 ϫ 105 pfu of PB2-Gluc, PB1-Gluc, or PA-Gluc recombinant virus. Enrichment Analysis—The integrated dataset of human protein– protein interactions (PPIs) available in the HIPPIE database was used to generate the subnetwork of PPIs connected as first neighbors to proteins directly targeted by the influenza virus polymerase subunits (layer one) This subset of PPIs connected to layer one was scored according to experimental evidence as described in HIPPIE [24]. Down-regulation of siRNA-targeted genes was evaluated at 48 h and 72 h post-transfection by means of RT-qPCR using the Maxima First Strand cDNA Synthesis Kit and Solaris qPCR Expression Assays and Master Mix (Thermo Scientific) according to the manufacturer’s recommendations
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