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

2013; Elsevier BV; Volume: 12; Issue: 10 Linguagem: Inglês

10.1074/mcp.m113.028688

1535-9484

Sandie Munier, Thomas Rolland, Cédric Diot, Yves Jacob, Nadia Naffakh,

Viral Infections and Immunology Research

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. 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. Influenza pandemics can be devastating. Even during typical epidemic years, ∼250,000 to 500,000 people worldwide die as a result of severe complications associated with influenza infections. The elucidation of how influenza viruses interact with the host cell machinery and hijack cellular pathways has become an active field of research and could pave the way to new antiviral approaches targeting virus–host interactions (1Shaw M.L. The host interactome of influenza virus presents new potential targets for antiviral drugs.Rev. Med. Virol. 2011; 21: 358-369Crossref PubMed Scopus (46) Google Scholar, 2Lee S.M. Yen H.L. Targeting the host or the virus: current and novel concepts for antiviral approaches against influenza virus infection.Antiviral Res. 2012; 96: 391-404Crossref PubMed Scopus (88) Google Scholar). Several RNAi-based genome-wide screens have been performed for the identification of host factors involved in influenza virus replication (3Watanabe T. Watanabe S. Kawaoka Y. Cellular networks involved in the influenza virus life cycle.Cell Host Microbe. 2010; 7: 427-439Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 4Stertz S. Shaw M.L. 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Because influenza virus polymerase is a key component in the viral life cycle and is a strong determinant of virulence and host range (10Naffakh N. Tomoiu A. Rameix-Welti M.A. van der Werf S. Host restriction of avian influenza viruses at the level of the ribonucleoproteins.Annu. Rev. Microbiol. 2008; 62: 403-424Crossref PubMed Scopus (175) Google Scholar, 11Resa-Infante P. Gabriel G. The nuclear import machinery is a determinant of influenza virus host adaptation.Bioessays. 2013; 35: 23-27Crossref PubMed Scopus (33) Google Scholar), other studies have used yeast two-hybrid (9Shapira S.D. Gat-Viks I. Shum B.O. Dricot A. de Grace M.M. Wu L. Gupta P.B. Hao T. Silver S.J. Root D.E. Hill D.E. Regev A. Hacohen N. A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection.Cell. 2009; 139: 1255-1267Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 12Tafforeau L. Chantier T. Pradezynski F. Pellet J. Mangeot P.E. Vidalain P.O. Andre P. 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Identification of cellular interaction partners of the influenza virus ribonucleoprotein complex and polymerase complex using proteomic-based approaches.J. Proteome Res. 2007; 6: 672-682Crossref PubMed Scopus (188) Google Scholar) to attempt to identify cellular proteins that interact with the viral polymerase. These approaches have limitations, as they do not recapitulate the entire viral cycle or cannot distinguish between direct and indirect interactions (16De Las Rivas J. Fontanillo C. Protein-protein interactions essentials: key concepts to building and analyzing interactome networks.PLoS Comput. Biol. 2010; 6: e1000807Crossref PubMed Scopus (433) Google Scholar). New technologies are thus needed to improve the mapping of virus–host interactions and to aid in the design of more effective therapeutics. The genome of influenza A viruses consists of eight molecules of single-stranded RNA of negative polarity. The viral RNAs associate with the nucleoprotein and with the three subunits of the polymerase complex (PB1, PB2, and PA) to form ribonucleoproteins (RNPs). 1The abbreviations used are:GOgene ontologyiPCAinfectious protein complementation assayLCSliterature-curated setMOImultiplicity of infectionMSmass spectrometryNLRnormalized luminescence ratioNPCnuclear pore complexpfuparticle forming unitPPIprotein–protein interactionRNPribonucleoproteinsRRSrandom reference setTPCKL-1-tosylamido-2-phenylethyl chloromethyl ketoneVSVvesicular stomatitis virus. 1The abbreviations used are:GOgene ontologyiPCAinfectious protein complementation assayLCSliterature-curated setMOImultiplicity of infectionMSmass spectrometryNLRnormalized luminescence ratioNPCnuclear pore complexpfuparticle forming unitPPIprotein–protein interactionRNPribonucleoproteinsRRSrandom reference setTPCKL-1-tosylamido-2-phenylethyl chloromethyl ketoneVSVvesicular stomatitis virus. Once in the infected cells, the RNPs are transported to the nucleus, where they undergo transcription and replication (17Resa-Infante P. Jorba N. Coloma R. Ortin J. The influenza virus RNA synthesis machine: advances in its structure and function.RNA Biol. 2011; 8: 207-215Crossref PubMed Google Scholar). We used a new strategy to detect binary interactions between the viral polymerase and host proteins during the course of an actual viral infection. Our infectious protein complementation assay (iPCA) is based on the complementation of two trans-complementing fragments of Gaussia princeps luciferase, Gluc1 and Gluc2 (18Cassonnet P. Rolloy C. Neveu G. Vidalain P.O. Chantier T. Pellet J. Jones L. Muller M. Demeret C. Gaud G. Vuillier F. Lotteau V. Tangy F. Favre M. Jacob Y. Benchmarking a luciferase complementation assay for detecting protein complexes.Nat. Methods. 2011; 8: 990-992Crossref PubMed Scopus (110) Google Scholar, 19Remy I. Michnick S.W. A highly sensitive protein-protein interaction assay based on Gaussia luciferase.Nat. Methods. 2006; 3: 977-979Crossref PubMed Scopus (348) Google Scholar). Interaction-mediated luciferase activity is measured in cultured cells transiently expressing a cellular protein fused to Gluc2 and infected with a recombinant influenza virus having Gluc1 fused to one of its polymerase subunits. These engineered infectious viruses allowed us to achieve the sensitive and accurate detection of influenza virus–host protein–protein interactions throughout the viral cycle. gene ontology infectious protein complementation assay literature-curated set multiplicity of infection mass spectrometry normalized luminescence ratio nuclear pore complex particle forming unit protein–protein interaction ribonucleoproteins random reference set L-1-tosylamido-2-phenylethyl chloromethyl ketone vesicular stomatitis virus. gene ontology infectious protein complementation assay literature-curated set multiplicity of infection mass spectrometry normalized luminescence ratio nuclear pore complex particle forming unit protein–protein interaction ribonucleoproteins random reference set L-1-tosylamido-2-phenylethyl chloromethyl ketone vesicular stomatitis virus. 293T, A549, and BSR cells were grown in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). MDCK cells were grown in modified Eagle's medium supplemented with 5% FCS. Wild-type A/WSN/33 (WSN) influenza virus was produced via reverse genetics using a procedure adapted from the work of Fodor et al. (20Fodor E. Devenish L. Engelhardt O.G. Palese P. Brownlee G.G. García-Sastre A. Rescue of influenza A virus from recombinant DNA.J. Virol. 1999; 73: 9679-9682Crossref PubMed Google Scholar). Vesicular stomatitis virus (VSV) (Indiana strain) was kindly provided by O. Delmas (Institut Pasteur, Paris, France). WSN and VSV viruses were amplified on MDCK and BSR cells, respectively, at a multiplicity of infection (MOI) of 0.001 pfu/cell. They were titrated on MDCK and BSR cells, respectively, using a plaque assay adapted from Matrosovich et al. (21Matrosovich M. Matrosovich T. Garten W. Klenk H.D. New low-viscosity overlay medium for viral plaque assays.Virol. J. 2006; 3: 63Crossref PubMed Scopus (356) Google Scholar). The eight pPolI plasmids containing the sequences corresponding to the genomic segments of influenza virus A/WSN/33 and the four recombinant pcDNA3.1 plasmids for the expression of WSN-PB1, -PB2, -PA, and -NP proteins (20Fodor E. Devenish L. Engelhardt O.G. Palese P. Brownlee G.G. García-Sastre A. Rescue of influenza A virus from recombinant DNA.J. Virol. 1999; 73: 9679-9682Crossref PubMed Google Scholar) were kindly provided by G. Brownlee (Sir William Dunn School of Pathology, Oxford, UK). The pPolI-SL-PB2-Gluc1 and pPolI-SL-PB2-Gluc2 plasmids were obtained by subcloning the Gluc1 and Gluc2 coding sequences, respectively, between the NotI and NheI sites of the pPolI-PB2-GFPS11 plasmid (22Avilov S.V. Moisy D. Munier S. Schraidt O. Naffakh N. Cusack S. Replication-competent influenza A virus that encodes a split-green fluorescent protein-tagged PB2 polymerase subunit allows live-cell imaging of the virus life cycle.J. Virol. 2012; 86: 1433-1448Crossref PubMed Scopus (67) Google Scholar). The pPolI-PB1-SL-Gluc1 and pPolI-PA-LL-Gluc1 plasmids were obtained via modification of the pPolI-WSN-PB1 and pPolI-WSN-PA using standard PCR and cloning procedures in order to fuse the following sequences to the 5′ end of the PB1- or PA-ORF: (a) a short sequence encoding the short peptidic linker AAAGGS (SL, for PB1) or the long peptidic linker AAAGGGGSGGGGS (LL, for PA); (b) the Gluc1 coding sequence; (c) a stop codon combined with an SpeI restriction site (TAAACTAGT); and (d) the 5′ terminal 88 nucleotides of the PB1 segment or the 5′ terminal 100 nucleotides of the PA segment. To produce the pPolI-PB1-SL-Gluc2 and pPolI-PA-LL-Gluc2 plasmids, the Gluc2 coding sequence was amplified using the pSPICA-N2 plasmid (18Cassonnet P. Rolloy C. Neveu G. Vidalain P.O. Chantier T. Pellet J. Jones L. Muller M. Demeret C. Gaud G. Vuillier F. Lotteau V. Tangy F. Favre M. Jacob Y. Benchmarking a luciferase complementation assay for detecting protein complexes.Nat. Methods. 2011; 8: 990-992Crossref PubMed Scopus (110) Google Scholar) as a template. The resulting PCR product was digested with NotI and NheI and subcloned between the NotI and SpeI sites of pPolI-PB1-Gluc1 and pPolI-PA-Gluc1 plasmids, respectively. The Gateway®-compatible donor plasmids containing cellular Open Reading Frames (ORFs) were obtained from the Human ORFeome resource (hORFeome v3.1). They were transferred into the Gateway®-compatible pSPICA-N2 destination vector (18Cassonnet P. Rolloy C. Neveu G. Vidalain P.O. Chantier T. Pellet J. Jones L. Muller M. Demeret C. Gaud G. Vuillier F. Lotteau V. Tangy F. Favre M. Jacob Y. Benchmarking a luciferase complementation assay for detecting protein complexes.Nat. Methods. 2011; 8: 990-992Crossref PubMed Scopus (110) Google Scholar) using LR clonase (Invitrogen) according to the manufacturer's specifications. The resulting plasmids allowed the expression of Gluc2-cellular-ORF fusion proteins. The proteins included in the random reference set (RRS) were randomly selected from within the RRS used by Braun et al. (23Braun P. Tasan M. Dreze M. Barrios-Rodiles M. Lemmens I. Yu H. Sahalie J.M. Murray R.R. Roncari L. de Smet A.S. Venkatesan K. Rual J.F. Vandenhaute J. Cusick M.E. Pawson T. Hill D.E. Tavernier J. Wrana J.L. Roth F.P. Vidal M. An experimentally derived confidence score for binary protein-protein interactions.Nat. Methods. 2009; 6: 91-97Crossref PubMed Scopus (334) Google Scholar). All constructs were verified through sequencing using a Big Dye terminator sequencing kit and an automated sequencer (PerkinElmer Life Sciences). The sequences of the oligonucleotides used for amplification and sequencing can be provided upon request. The method used for the production of recombinant influenza viruses via reverse genetics was adapted from previously described procedures (20Fodor E. Devenish L. Engelhardt O.G. Palese P. Brownlee G.G. García-Sastre A. Rescue of influenza A virus from recombinant DNA.J. Virol. 1999; 73: 9679-9682Crossref PubMed Google Scholar). Briefly, the eight pPolI-WSN and four pcDNA3.1-WSN plasmids (0.5 μg of each) were co-transfected into a sub-confluent monolayer of co-cultivated 293T and MDCK cells (4 × 105 and 3 × 105 cells, respectively, seeded in a 35-mm dish) using 10 μl of FuGENE® HD transfection reagent (Roche). After 24 h of incubation at 35 °C, the supernatant was removed, and cells were washed twice with DMEM and incubated at 35 °C in DMEM containing TPCK-treated trypsin at a final concentration of 1 μg/ml for 48 h. The efficiency of reverse genetics was evaluated by titrating the supernatant on MDCK cells in plaque assays. For subsequent amplification of the recombinant PB1-, PB2-, and PA-Gluc1 or PB1-, PB2-, and PA-Gluc2 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 μg/ml. Viral stocks were titrated via plaque assays on MDCK cells as described elsewhere (21Matrosovich M. Matrosovich T. Garten W. Klenk H.D. New low-viscosity overlay medium for viral plaque assays.Virol. J. 2006; 3: 63Crossref PubMed Scopus (356) Google Scholar). Viral RNA was extracted and subjected to reverse-transcription and amplification using specific oligonucleotides. The products of amplification were purified using a QIAquick gel extraction kit (Qiagen, Courtaboeuf, France) and were sequenced using a Big Dye terminator sequencing kit and an automated sequencer (PerkinElmer Life Sciences). The sequences of the oligonucleotides used for amplification and sequencing can be provided upon request. MDCK cells were seeded at a concentration of 2 × 104 cells per well in 96-well white plates (Greiner Bio-One, Courtaboeuf, France). After 24 h, cells were rinsed twice with DMEM and co-infected with a combination of vP-Gluc1 and vP′-Gluc2 viruses (0.01 pfu of each virus/cell in 50 μl). After viral adsorption for 1 h at 35 °C, 50 μl of DMEM supplemented with 4% of FCS were added and cells were incubated at 35 °C for 24 h. Cells were either left untreated or incubated with ribavirin (Sigma) or nucleozin (Sigma) at a final concentration of 1–100 μm or 0.01–1 μm, respectively, for 24 h post-infection, or they were incubated with amantadine (Sigma) at a final concentration of 10 μm during the 30 min preceding infection, during viral adsorption, and for 24 h post-infection. At 24 h post-infection, cells were mildly rinsed in Ca2+/Mg2+-Dulbecco's PBS, and 40 μl of Renilla lysis buffer (Promega, Charbonnières, France) were directly added to each well. After incubation for 1 h at room temperature, the Gaussia princeps luciferase enzymatic activity was measured using the Renilla luciferase assay reagent (Promega) and a Berthold Centro XS luminometer (Renilla luminescence counting program, integration time of 10 s after injection of 50 μl of the reagent). 293T cells were seeded at a concentration of 3 × 104 cells per well in 96-well white plates (Greiner Bio-One, Courtaboeuf, France). After 24 h, the cells were co-transfected in triplicate with 100 ng of the recombinant p-Gluc2-cellular-ORF constructs and 50 ng of the pCI plasmid (Promega) using polyethyleneimine (Polysciences Inc, Le Perray-en-Yvelines, France). At 17 h post-transfection, cells were rinsed with DMEM and infected with 50 μl of a viral suspension containing 9 × 105 pfu of PB2-Gluc1, PB1-Gluc1, or PA-Gluc1 recombinant virus. As a control, cells were either (i) co-transfected with 100 ng of the empty p-Gluc2 plasmid and 50 ng of pCI and subsequently infected with 9 × 105 pfu of PB2-Gluc1, PB1-Gluc1, or PA-Gluc1 recombinant virus or (ii) co-transfected with 100 ng of the p-Gluc2-cellular-ORF constructs and 50 ng of the empty p-Gluc1 plasmid and subsequently infected with 9 × 105 pfu of the wild-type WSN virus. After viral adsorption for 1 h at 35 °C, 50 μl of DMEM supplemented with 4% FCS were added to each well, and cells were incubated at 35 °C for 6 h. Cells were mildly rinsed in Ca2+/Mg2+-Dulbecco's PBS, and 40 μl of Renilla lysis buffer (Promega) were directly added to each well. After incubation for 1 h at room temperature, the Gaussia princeps luciferase enzymatic activity was measured using the Renilla luciferase assay reagent (Promega) and a Berthold Centro XS luminometer (Renilla luminescence counting program, integration time of 2 s after injection of 50 μl of the reagent). For each p-Gluc2-cellular-ORF plasmid, three normalized luminescence ratios (NLRs) were calculated as follows: the luminescence activity measured in cells transfected with the p-Gluc2-cellular-ORF plasmid and infected with the PB2-Gluc1, PB1-Gluc1, or PA-Gluc1 virus (arbitrary units, mean of triplicates) was divided by the sum of the luminescence activities measured in both control samples as described above (arbitrary units, mean of triplicates). 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 (24Schaefer M.H. Fontaine J.F. Vinayagam A. Porras P. Wanker E.E. Andrade-Navarro M.A. HIPPIE: integrating protein interaction networks with experiment based quality scores.PLoS One. 2012; 7: e31826Crossref PubMed Scopus (237) Google Scholar). Interactions with scores greater than or equal to 0.75, considered as high-confidence interactions, were selected as layer two. The resulting subnetwork was analyzed with ClueGO (Cytoscape plugin (25Bindea G. Mlecnik B. Hackl H. Charoentong P. Tosolini M. Kirilovsky A. Fridman W.H. Pages F. Trajanoski Z. Galon J. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks.Bioinformatics. 2009; 25: 1091-1093Crossref PubMed Scopus (3819) Google Scholar)) to extract the non-redundant biological information based on enrichment for biological process descriptors in gene ontology (GO) annotations associated with each polymerase subunit. The significance of each term was calculated using a hypergeometric test and a Bonferroni p value correction according to ClueGO standard statistical options (25Bindea G. Mlecnik B. Hackl H. Charoentong P. Tosolini M. Kirilovsky A. Fridman W.H. Pages F. Trajanoski Z. Galon J. ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks.Bioinformatics. 2009; 25: 1091-1093Crossref PubMed Scopus (3819) Google Scholar). GO term analysis was restricted to p values ≤ 0.01 and "global" network predefined selection criteria in order to get more global functional enrichment information and obtain insights into the main biological processes susceptible to targeting by the influenza virus polymerase subunits. Small interfering RNAs (siRNAs) targeting CCT2, RANGAP1, and NUP62 were purchased from Thermo Scientific (Illkirch, France). Non-target siRNA (Dharmacon ON-TARGETplus Non-targeting Control pool) was used as a negative control. A549 cells (2 × 104 cells) were transfected in suspension with 25 nm of siRNA using 0.2 μl of DharmaFECT1 transfection reagent (Thermo Scientific) and were plated in 96-well plates. The medium was replaced with fresh medium 24 h later. At 48 h post-transfection, cells were infected with influenza A/WSN/33 virus at an MOI of 0.001 pfu/cell or with VSV at an MOI of 0.0001 pfu/cell. After viral adsorption for 1 h at 37 °C, 50 μl of DMEM supplemented with 4% FCS were added per well and cells were incubated at 37 °C for 24 h. Supernatants were collected and viral titers were determined via plaque assays on MDCK or BSR cells as described above. Cell viability was determined by assaying the total intracellular ATP with the CellTiter-Glo Luminescent Viability Assay kit (Promega) according to the manufacturer's recommendations. 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. Using a plasmid-based reverse genetics system (20Fodor E. Devenish L. Engelhardt O.G. Palese P. Brownlee G.G. García-Sastre A. Rescue of influenza A virus from recombinant DNA.J. Virol. 1999; 73: 9679-9682Crossref PubMed Google Scholar, 26Dos Santos Afonso E. Escriou N. Leclercq I. van der Werf S. Naffakh N. The generation of recombinant influenza A viruses expressing a PB2 fusion protein requires the conservation of a packaging signal overlapping the coding and noncoding regions at the 5′ end of the PB2 segment.Virology. 2005; 341: 34-46Crossref PubMed Scopus (67) Google Scholar, 27Neumann G. Kawaoka Y. Genetic engineering of influenza and other negative-strand RNA viruses containing segmented genomes.Adv. Virus Res. 1999; 53: 265-300Crossref PubMed Scopus (27) Google Scholar), we produced a set of infectious recombinant A/WSN/33 (WSN) influenza viruses that expressed a PB1, PB2, or PA polymerase subunit fused at their C-terminal end to Gluc1 or Gluc2 complementation fragments of Gaussia princeps luciferase. Viability of the recombinant viruses depended on the peptidic linker separating the Gluc fragment from the polymerase subunits, and on sequence duplication ensuring the conservation of packaging signals at the 5′ end of the viral genomic segments (26Dos Santos Afonso E. Escriou N. Leclercq I. van der Werf S. Naffakh N. The generation of recombinant influenza A viruses expressing a PB2 fusion protein requires the conservation of a packaging signal overlapping the coding and noncoding regions at the 5′ end of the PB2 segment.Virology. 2005; 341: 34-46Crossref PubMed Scopus (67) Google Scholar) (Fig. 1). Despite moderate attenuation relative to the wild-type WSN virus, the viruses expressing a viral fusion protein (vP-Gluc1 or vP-Gluc2) were replication-competent, with the vP-Gluc1 viruses showing higher titers than their vP-Gluc2 counterparts upon multi-cycle amplification on MDCK cells (2 × 106 to 10 × 106 pfu/ml, compared with 0.3 × 106 to 3 × 106 pfu/ml). We checked whether the viral fusion proteins were indeed expressed in vP-Gluc1 and vP-Gluc2 infected cells, and whether they could participate in the reconstitution of a functional Gaussia princeps luciferase. To this end, MDCK cells were infected with six distinct pairs corresponding to every possible combination of vP-Gluc1 and vP-Gluc2 viruses. High luciferase activities were detected in cell lysates at 24 h post-infection (Fig. 2A, black bars), which likely resulted from the natural assembly of viral polymerase heterotrimers. A strong and dose-dependent reduction of luciferase activities was observed in the presence of ribavirin (Fig. 2A, white bars, and Fig. 2B) and in the presence of nucleozin (Fig. 2C), but not in the presence of amantadine (Fig. 2A, grey bars), consistent with the A/WSN/33 virus used in these experiments being sensitive to ribavirin and nucleozin but resistant to amantadine (28Sidwell R.W. Bailey K.W. Wong M.H. Barnard D.L. Smee D.F. In vitro and in vivo influenza virus-inhibitory effects of viramidine.Antiviral Res. 2005; 68: 10-17Crossref PubMed Scopus (105) Google Scholar, 29Takeda M. Pekosz A. Shuck K. Pinto L.H. Lamb R.A. Influenza a virus M2 ion channel activity is essential for efficient replication in tissue culture.J. Virol. 2002; 76: 1391-1399Crossref PubMed Scopus (207) Google Scholar). The observed EC50 values for ribavirin and nucleozin were 9.7 μm and 0.1