Exploration of Binary Virus–Host Interactions Using an Infectious Protein Complementation Assay

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 transcomplementation 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. Molecular & Cellular

Systems-Level Analysis of Innate Immunity

“…PD1 prevented viral RNA export from the nucleus, a crucial step in viral replication, by disrupting viral RNA binding to NXF1, an mRNA transporter. This observation is supported by two high-throughput studies: A genome-wide RNAi screen showed that NXF1i sessential for viral replication (92), and a proteomic study identified it as a cellular interacting partner of the influenza viral polymerase complex (93). …”

Targeting Viral Proteostasis Limits Influenza Virus, HIV, and Dengue Virus Infection

“…As such, a molecular understanding of how each viral protein co-opts and interferes with cellular processes during infection is critical for the elucidation of mechanisms of pathogenesis and for the development of novel therapeutic strategies. To gain insight into virus-host protein interactions, studies have been performed using yeast 2-hybrid systems, complementation assays, or affinity purification of epitope tagged viral proteins transfected into cells (Bradel-Tretheway et al, 2011; de Chassey et al, 2013; Gorai et al, 2012; Guan et al, 2012; Jorba et al, 2008; Lin et al, 2012; Mayer et al, 2007; Munier et al, 2013; Ngamurulert et al, 2009; Shapira et al, 2009; Tafforeau et al, 2011). Although these approaches have indicated potential cellular factors targeted by viral proteins, they are limited by the fact that the mere expression of influenza proteins does not recapitulate key physiological aspects of the host-virus battleground.…”

Temperature-Sensitive Mutants in the Influenza A Virus RNA Polymerase: Alterations in the PA Linker Reduce Nuclear Targeting of the PB1-PA Dimer and Result in Viral Attenuation

“…The pCI-PA-LL-Gluc1 plasmid and its derivatives (pCI-PA214P-Gluc1, pCI-PA216P-Gluc1, pCI-PA219P-Gluc1, and pCI-PA223P-Gluc1) and pCI-PB1-SL-Gluc2 plasmids were obtained via modification of pCI-PA and pCI-PB1 by using standard PCR, site-directed mutagenesis, and cloning procedures in order to fuse the following sequences to the 3= end of the PA ORFs and PB1 ORF: a long peptidic linker, AAAGGGGSGGGGS (LL), followed by Gluc1 for PA and a short peptidic linker, AAAGGS (SL), followed by Gluc2 for PB1. The Gateway-derived Gluc2-IPO5 expression plasmid was described previously (34). 293T cells were seeded at a concentration of 3 ϫ 10 4 cells per well in 96-well white plates (Greiner Bio-One, Courtaboeuf, France).…”

“…The deficiency of PA-PB1 targeting the nucleus at nonpermissive temperatures may be due to instability of the PA-PB1 complex and/or to inefficient binding to the ␤-importin IPO5, previously shown to be involved in the nuclear import of the PA-PB1 complex (29,38). To investigate these hypotheses, we used a Gaussia princeps luciferase-based complementation assay (33,34) to quantify the binding of PA mutants to PB1 at permissive (35°C) and nonpermissive (39.5°C) temperatures. Figure 8 shows that all the mutations tested (L214P, D216P, L219P, and F223P) impaired the binding with PB1 at both temperatures and that this defect was more pronounced at 39.5°C than at 35°C.…”