Published Ahead of Print 12 October 2011.
2011, 85(24):13010. DOI: 10.1128/JVI.02651-10.
Patrice Andre, Chantal Rabourdin-Combe and Vincent
Johann Pellet, Philippe E. Mangeot, Pierre-Olivier Vidalain,
Lionel Tafforeau, Thibault Chantier, Fabrine Pradezynski,
an Influenza Virus Polymerase Cellular
Generation and Comprehensive Analysis of
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JOURNAL OF VIROLOGY, Dec. 2011, p. 13010–13018
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 24
Generation and Comprehensive Analysis of an Influenza
Virus Polymerase Cellular Interaction Network?†§
Lionel Tafforeau,1,2* Thibault Chantier,1,2Fabrine Pradezynski,1,2Johann Pellet,1,2
Philippe E. Mangeot,1,2Pierre-Olivier Vidalain,3,4Patrice Andre,1,2,5
Chantal Rabourdin-Combe,1,2and Vincent Lotteau1,2,5
Universite ´ de Lyon, Lyon, France1; INSERM U851, Lyon, France2; Institut Pasteur, Paris, France3; Unité de Genomique Virate et
Vaccination, CNRS URA3015, Paris, France4; and Laboratoire de Virologie, Hospices Civils de Lyon, Lyon, France5
Received 21 December 2010/Accepted 28 September 2011
The influenza virus transcribes and replicates its genome inside the nucleus of infected cells. Both activities
are performed by the viral RNA-dependent RNA polymerase that is composed of the three subunits PA, PB1,
and PB2, and recent studies have shown that it requires host cell factors to transcribe and replicate the viral
genome. To identify these cellular partners, we generated a comprehensive physical interaction map between
each polymerase subunit and the host cellular proteome. A total of 109 human interactors were identified by
yeast two-hybrid screens, whereas 90 were retrieved by literature mining. We built the FluPol interactome
network composed of the influenza virus polymerase (PA, PB1, and PB2) and the nucleoprotein NP and 234
human proteins that are connected through 279 viral-cellular protein interactions. Analysis of this interactome
map revealed enriched cellular functions associated with the influenza virus polymerase, including host factors
involved in RNA polymerase II-dependent transcription and mRNA processing. We confirmed that eight
influenza virus polymerase-interacting proteins are required for virus replication and transcriptional activity
of the viral polymerase. These are involved in cellular transcription (C14orf166, COPS5, MNAT1, NMI, and
POLR2A), translation (EIF3S6IP), nuclear transport (NUP54), and DNA repair (FANCG). Conversely, we
identified PRKRA, which acts as an inhibitor of the viral polymerase transcriptional activity and thus is
required for the cellular antiviral response.
Influenza A virus is responsible for annual epidemics of
respiratory disease and reoccurring pandemics and represents
an important public health problem worldwide. Its genome is
composed of eight single-stranded negative-polarity viral RNA
(vRNA) segments. They are individually encapsidated by nu-
cleoprotein (NP) and the RNA-dependent RNA polymerase
(RdRP), forming a viral ribonucleoprotein (vRNP) complex
(reviewed in reference 38). Each vRNA behaves as an inde-
pendent template for transcription and replication that are
both taking place in the nucleus of infected cells. The virus
RNA polymerase is a heterotrimer composed of subunits PA,
PB1, and PB2. PB1 is the core subunit of the complex and
contains the polymerase activity, while PB2 recognizes capped
cellular mRNA (38) and PA possesses an endonuclease activity
(12). After viral decapsidation, vRNPs are transported into the
nucleus, where they are engaged in primary transcription. This
is initiated by cap snatching of cellular pre-mRNA: the PB2
subunit recognizes capped mRNAs, while the PA subunit
cleaves them 10 to 15 nucleotides (nt) downstream of the cap,
generating cap-containing primers for virus mRNA synthesis
(28). Viral mRNAs are then translated by cytoplasmic ribo-
somes, allowing newly synthesized components of the viral
polymerase and NP to accumulate in the nucleus. It has re-
cently been proposed that the nuclear accumulation of newly
synthesized viral proteins could be responsible for the switch
from viral transcription to replication (56). For the genome
replication, untranslated sequences at the 5? and the 3? ends of
each genomic vRNA segment act as promoter elements that
are recognized by the viral polymerase. vRNA segments are
copied into positive-strand complementary RNAs (cRNAs),
which are encapsidated by NP and serve as templates for
de novo vRNA synthesis (22, 38).
The influenza virus polymerase performs numerous func-
tions during the virus life cycle, suggesting that many cellular
factors interact with this complex and are required for the viral
genome’s transcription and replication.
Only a few cellular partners have been previously described
in the literature, but recent studies identified several influenza
virus polymerase interactors using proteomic approaches (23,
33). Moreover, a global survey of influenza virus host cell
partners was established using the yeast two-hybrid (Y2H)
technology (51). A series of functional genome-wide small
interfering RNA (siRNA) screenings were also conducted for
the identification of host factors involved in influenza virus
replication (5, 18, 24, 27, 51; reviewed in reference 58). We
present here a more specific analysis that is focused on the
RdRP cellular interactors. We reconstructed a global influenza
virus polymerase physical interaction map by performing yeast
two-hybrid screens with each subunit as bait and by retrieving
protein-protein interactions from the literature. Through this
interaction network, we identified cellular functions specifically
* Corresponding author. Present address: RNA Metabolism,
IBMM, ULB, rue Profs. Jeener & Brachet 12, 6041 Gosselies, Bel-
gium. Phone: 32 26 50 97 74. Fax: 32 26 50 97 47. E-mail: lionel
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 12 October 2011.
§ The authors have paid a fee to allow immediate free access to
on November 21, 2011 by guest
targeted by the viral polymerase. We selected nine functionally
relevant host cell partners, whose interactions with the viral
proteins were validated in human cells. We show that these
cellular factors affect both the viral polymerase activity and the
virus replication using functional assays: eight of them are
required for virus replication and polymerase activity. Con-
versely, PRKRA acts as an antiviral factor since virus replica-
tion and polymerase activity are enhanced when its expression
MATERIALS AND METHODS
Cloning of influenza virus ORFs. NP, PA, PB1, and PB2 open reading frames
(ORFs) from A/PR/8/34 (H1N1) and A/WSN/33 (H1N1) (kindly provided by G.
Brownlee and V. Moule `s, respectively), PA and PB1 from A/VietNam/1194/2004
(H5N1) (kindly provided by V. Moule `s), PA from A/Turkey/651242/2006
(H5N1) (the H5N1 genomic RNAs were kindly provided by V. Moule `s), and PB2
from A/Victoria/3/75 (H3N2) (kindly provided by D. Hart) were amplified from
plasmids encoding corresponding cDNA genomic segments or from genomic
viral RNA by using ORF-specific Gateway primers (containing attB1.1 at the 5?
end and attB2.1 at the 3? end and without ATG and stop codons ). Four PB2
fragments isolated from the ESPRIT technology were screened: the long (amino
acids [aa] 234 to 496) and the short (aa 318 to 483) (17) cap-binding domains,
the “627” domain (aa 538 to 693), encompassing the K627 residue (55), and the
C-terminus domain (CTD) (DPDE; aa 678 to 759) (54). We constructed the
following compensatory point mutation mutants: K627E in the 627 domain (52)
and D701N (49), R702K (15), and the D701N R702K double-point mutant in the
DPDE domain. After PCR, ORFs were cloned by in vitro recombination into
donor vectors (pDONR207/223). All clones were sequence verified and stored in
the viralORFeotheque repository. We developed a database that provides an
integrated set of bioinformatic tools to clone viral ORFs in the Gateway system,
viralORFeome (http://www.viralorfeome.com ).
Y2H assay. Viral ORFs were cloned by recombination into pGBKT7-gw and
transformed in AH109 (bait strain; Clontech). Y2H screens were performed by
yeast mating (53). Briefly, the human cDNA libraries (comprising spleen, fetal
brain, and respiratory epithelium libraries from Invitrogen and customized with
CloneMiner for the latter) were transformed in Y187 (prey strain; Clontech),
and each bait strain was mated with the library prey strain. Diploids were plated
on synthetic defined medium lacking tryptophan, leucine, and histidine and
supplemented with 3-aminotriazole (SD?W?L?H ?3-AT), and positive clones
were streaked twice on this selection medium. AD-cDNAs (where AD refers to
the Gal4 activation domain fused to cDNA) were PCR amplified and se-
quenced. Interaction sequence tags were analyzed through pISTil and depos-
ited in the viralORFeome database (42). All partners of PB2 domains identified in
Y2H screens were retested against each PB2 fragment in a Y2H array system (in
an all-against-all matrix). First, AD-cDNAs encoding cellular interactors identi-
fied with PB2 domains were transformed in the yeast prey strain together with
linearized pACT2-gw. Bait and prey strains were arrayed in a 96-well format
using a robotic workstation (Tecan Freedom Evo) and mated in an all-against-all
array on YPD plates. Diploids were selected on SD ?W ?L for 2 days, and then
transferred on selection medium (SD ?W ?L ?H plus increasing concentra-
tions of 3-AT). Interactions were scored as positive if observed at least twice in
3 independent arrays (53). Among the 79 partners identified with PB2 domains,
59 were positively scored, confirming the results obtained during the screen. The
20 remaining partners were discarded.
GO category enrichment using BiNGO and Golorize on Cytoscape. BiNGO
(Biological Network Gene Ontology) is a Cytoscape plugin that assesses which
Gene Ontology (GO) categories are overrepresented in a network (32). It pro-
vides single P values, calculated with the hypergeometric test, and takes into
consideration both the total number of genes from the analyzed data set and the
total number of genes that are linked to the same ontology term, as well as
multiple testing of corrected P values, calculated using the Benjamini and
Hochberg false discovery rate (FDR) correction, for the enrichment of each GO
term (in our case, “biological process”). GOlorize is a Cytoscape plugin that
highlights the class members of the enriched categories identified by BiNGO
using a color code within the Cytoscape-built network (16, 50). The cross-
validation of our Y2H interaction data set revealed a false-positive rate of 15%.
To take into account of this rate in the functional enrichment analysis, we
generated 50 different clusters of 93 randomly chosen Y2H interactors (repre-
senting 85% of the 109 Y2H interactors) plus all of the cellular partners in the
literature. For each cluster, we performed a GO analysis using BiNGO. We
considered positive the overrepresented classes that were scored positive for all
the 50 clusters. Nine functional categories were selected, and the class members
were colorized within the FluPol network using GOlorize. Note that we did not
change the network layout after the GOlorize analysis.
FluPol network visualization. We used Cytoscape Web API for visualizing and
manipulating the FluPol network graph, allowing a dynamic network display (31;
http://flupol.lyon.inserm.fr). All of the interaction data can be exported through
this web page.
BiFC. For the bifunctional fluorescence complementation (BiFC) assay,
A/WSN/33 PA, PB1, and PB2 were cloned into Polyc vector, where they were
fused upstream of the C-terminal moiety of yellow fluorescent protein (YFP).
The cellular partners’ genes were amplified from the human cDNA Y2H librar-
ies and then in vitro cloned into pDONR207 as viral ORFs (see above), or were
retrieved from the hORFeome collection (48). They were then cloned by recom-
bination in pGWEN, in fusion downstream of the N-terminal moiety of Venus
(modified YFP) (26). HEK-293T cells were cotransfected using JetPei (Polyplus)
with each combination of plasmid pair in a 96-well plate. Forty-eight hours after
transfection, cells were resuspended in 100 ?l phosphate-buffered saline (PBS),
and YFP signal was measured using a FACSArray (BD). The percentage of
fluorescent cells was measured (mean of three independent experiments), and an
interaction was considered positive when more than 5% of cells were fluorescent,
according to results obtained with negative controls.
FCPI assay. The flow cytometry protein interaction (FCPI) assay was set up
using two couples of interacting proteins (see Fig. S1 in the supplemental ma-
terial). Viral ORFs (A/WSN/33 strain) were cloned by in vitro recombination into
pCMV-BioEase-Cherry-gw, where they are fused downstream of the BioEase tag
(Invitrogen) and the fluorescent protein mCherry. Cellular genes were trans-
ferred to pCMV-eGFP-gw (kindly provided by Y. Jacob), to be fused down-
stream to enhanced green fluorescent protein (EGFP). Human HEK-293T cells
were cotransfected using JetPei (Polyplus) with each combination of plasmid pair
(1 ?g of each vector) in a 12-well plate and lysed 48 h after transfection. Sixty
micrograms of protein extracts was suspended in 200 ?l lysis buffer (150 mM
NaCl, 20 mM Tris-HCl [pH 8], 1 mM EDTA, 0.5% NP-40 plus Complete
miniprotease inhibitor cocktail [Roche]) containing 1 ?l of streptavidin-conju-
gated microspheres (Polysciences) on a multiwell filter plate (Pall) and incubated
for 2 h at 4°C under gentle agitation. Each affinity purification was performed in
triplicate. The beads were washed twice with 200 ?l of wash buffer (same as lysis
buffer, without the protease inhibitor cocktail) using a vacuum manifold and then
were resuspended in 100 ?l of wash buffer, and bead-associated GFP signal was
analyzed using a FACSArray (BD). The analysis of results is based on the
relative mean fluorescence intensity (MFI) associated with the beads compared
to the MFI of an empty bait vector. An interaction is considered positive when
the difference is higher than 5%.
Cells and viruses. A549 and HEK-293T cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin and
10% fetal bovine serum at 37°C and 5% CO2. Influenza A virus A/New Cale-
donia/2006 (H1N1) (clinical isolate) was propagated in MDCK cells. Titers of
viral stocks were determined by PFU assay on MDCK cells.
Coimmunoprecipitation and protein detection. Cellular genes were trans-
ferred to pCIneo-3Flag-gw (kindly provided by Y. Jacob), to be fused down-
stream to 3? Flag epitope (Sigma). A549 cells were transfected in 6-well plates
using Turbofect (Fermentas). Twenty-four hours after transfection, cells were
infected by H1N1 A/New Caledonia/2006 virus at a multiplicity of infection
(MOI) of 1 and lysed 24 h after infection. Three hundred micrograms of protein
extracts was suspended in 1 ml lysis buffer containing 20 ?l of anti-Flag M2
magnetic beads (Sigma) and incubated for 2 h at 4°C under gentle agitation. The
beads were washed three times with 1 ml of wash buffer on a magnetic rack, and
then precipitates were eluted in 20 ?l SDS loading buffer without reducing
agents and boiled for 5 min at 95°C. Total cell extracts (20 ?g) and immuno-
precipitates were separated by SDS-PAGE and analyzed by Western blotting
using antibodies against PB1 (rabbit polyclonal antibody kindly provided by
J. Ortin) and Flag (horseradish peroxidase [HRP]-conjugated mouse monoclonal
antibody; Sigma). The secondary anti-rabbit antibody was also HRP conjugated
(Santa Cruz). EGFP-PRKRA was detected with mouse monoclonal anti-GFP
Influenza virus replication assay and virus titer determination using
MUNANA. Human A549 cells were transfected with siRNA targeting each gene
(using a pool of 2 siRNAs at a concentration of 40 nM; Invitrogen) in a reverse
transfection procedure with Lipofectamine RNAiMAX (Invitrogen) in a 96-well
plate. Forty-eight hours after siRNA transfection, cells were infected by H1N1
A/New Caledonia/2006 virus at an MOI of 0.5 in DMEM containing 1% peni-
cillin/streptomycin and 0.25 ?g/ml TPCK (tosylsulfonyl phenylalanyl chlorom-
ethyl ketone)-treated trypsin (Sigma). Cell culture supernatants (25 ?l) were
VOL. 85, 2011 HOST CELL PARTNERS OF THE INFLUENZA VIRUS POLYMERASE13011
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collected at 24 h and 48 h postinfection, and virus titers were determined by
quantifying the neuraminidase activity using a MUNANA [2?-(4-methylumbel-
liferyl)-?-D-N-acetylneuraminic acid] assay, as described in reference 59, with
minor modifications. Briefly, cell supernatants were transferred into a 96-well
black flat-bottom plate and mixed with 25 ?l of PBS with Ca2?/Mg2?(Invitro-
gen) and 50 ?l of MUNANA stock solution (20 ?M; Sigma). Plates were
incubated 1 h at 37°C, and the reaction was stopped by adding 100 ?l of Stop
solution (glycine 0.1 M [pH 10.7], 25% ethanol). The amount of fluorescent
product released by MUNANA hydrolysis (4-methylumbelliferyl [4-MU]) was
measured in a Tecan spectrophotometer with excitation and emission wave-
lengths of 365 and 450 nm, respectively. A reference curve was established in
parallel with serial dilutions of a titrated stock of influenza virus.
To assess potential cellular toxicity induced by siRNAs, a cell viability assay
was performed using the resazurin-based fluorometric assay at 48 h posttrans-
fection (see Fig. 2C). Resazurin (1 mg/ml of medium; Sigma) detects cell viability
by converting to a red fluorescent dye in response to reduction of growth medium
resulting from cell growth. The fluorescent signal generated is proportional to
the number of living cells. After an incubation for 2 h at 37°C, the fluorescence
was measured with a Tecan spectrophotometer with excitation and emission
wavelengths of 550 and 590 nm, respectively. A reference curve was established
with known serial dilutions of growing cells.
Influenza virus minigenome replicon assay. (i) Overexpression of human
partner proteins. Human HEK-293T cells (plated in a 96-well plate) were
cotransfected using JetPei (Polyplus) with a combination of plasmids expressing
a cellular partner (100 ng), vectors encoding the A/WSN/33 polymerase subunits
(PA, PB1, and PB2) and NP (50 ng each plasmid), a reporter plasmid (pPOLI-
Luc-RT, encoding the firefly luciferase in the negative-sense orientation flanked
by the noncoding regions of the segment 8 of A/WSN/33, driven by a polymerase
I [Pol I] promoter, kindly provided by M. Shaw ; 50 ng), and a vector
constitutively expressing Renilla luciferase (5 ng pRL-SV40-Rluc; Promega).
Twenty-four hours and 48 h after transfection, luciferase activities were deter-
mined using the Dual-Glo luciferase assay system (Promega). As a positive
control, cells were transfected by the complete set of vRNP and an empty cellular
protein expression vector. As a negative control, cells were transfected with the
same reaction mixture but without the plasmid expressing PB1.
(ii) Depletion of human partner genes. Human HEK-293T cells were trans-
fected with siRNA targeting each gene as described above. Knockdown was
allowed to proceed for 48 h, and then cells were transfected with the complete set
of vRNP components (plasmids expressing NP, PA, PB1, and PB2 and the
reporter vector encoding firefly luciferase) as described above. As positive con-
trol, cells were transfected by a scrambled control siRNA, and then with the
complete set of the vRNP components. As a negative control, cells were treated
with the same mixture but without PB1-expressing plasmid.
RESULTS AND DISCUSSION
Building an influenza virus polymerase cellular interactome
network. We generated a comprehensive protein-protein in-
teraction network between the viral influenza A virus polymer-
ase (FluPol) and its host cell partners using the yeast two-
hybrid (Y2H) system. For this purpose, human cDNA libraries
were screened with the RdRP subunits and NP from different
virus strains (H1N1, H3N2, and H5N1). PB2 fragments that
were isolated by the ESPRIT technology and of which struc-
tures were defined were also screened individually (17, 54, 55,
60). Human cDNA libraries from three different tissues
(spleen, fetal brain, and respiratory epithelium) were used,
covering a wide range of the human proteome as well as a
tissue that is specifically targeted by the influenza A virus.
Altogether, we discovered 112 viral-human protein interac-
tions involving 109 human proteins, including 5 interactions
with NP, 30 with PA, 18 with PB1, and 59 with PB2. Among
them, 3 human proteins interact both with PA and PB2 (see
Table S1 in the supplemental material and Fig. 1A and 1B).
To assess the confidence of the yeast two-hybrid results, two
validation methods in human cells were used. Thirty-four cel-
lular partners (31% of the Y2H interactors) were randomly
retested for their interaction with the viral protein bait that
trapped them in the Y2H assay by either bifunctional fluores-
cence complementation (BiFC) (26) or a flow cytometric
method developed in our lab to measure protein-protein in-
teractions (FCPIA, similar to the assay developed by the
Neubig lab ). While the bait (in our case, the viral protein) is
fused to both the BioEase tag, an in vivo biotinylated sequence
that can be trapped using streptavidin beads, and to the fluo-
rescent protein mCherry (allowing its detection on beads), the
prey is fused to eGFP. This assay allows detection of copurified
preys on streptavidin beads using a flow cytometer (Materials
and Methods; see Fig. S1 in the supplemental material).
KPNA1 was used as positive control as it interacts with PB2
and NP (39, 54). Among the 34 host cell partners tested, 29
were validated using either BiFC, FCPIA, or both assays (in
green in Table 1; see Table S5 in the supplemental material).
We identified 18 new connections through these validation
assays, by performing an array in which the cellular proteins
were a priori tested against different viral baits (in blue in Table
1). It is of note that we did not validated the interactions
identified in the Y2H assay for three cell partners but discov-
TABLE 1. List of consolidated FluPol interactorsa
aGreen, interaction found in Y2H and validated in either BiFC or FCPIA;
yellow, interaction that was tested either with BiFC or FCPIA but was negative;
blue, novel interaction detected in either BiFC or FCPIA. Raw values are given
in Table S5 in the supplemental material.
bThe peGFP-empty vector (eGFP/) was used as a negative control in FCPIA.
cThe pGwen-empty vector (Gwen/) was used as a negative control in BiFC.
13012 TAFFOREAU ET AL.J. VIROL.
on November 21, 2011 by guest
ered interactions with different viral proteins (EIF3S6IP,
FANCG, and ZCCHC17). Twenty-nine of the 34 Y2H inter-
actors in the subset tested were validated, giving a false-posi-
tive rate of 15%, which is in accordance with previous studies
(9). Most of the cellular proteins interact with one FluPol
subunit (17 of the 29 validated), like C14orf166, which only
interacts with PA, or EEF1A1, which only interacts with PB1.
We also observed that a few host cell partners interact with
both PB1 and PB2 (the 5 cellular proteins CES1, CHAF1A,
DDX54, MNAT1, and NMI). PCNA and PSMA7 interact with
the FluPol subunits (PA, PB1, and PB2), whereas EEF1D
interacts with all of the vRNP protein constituents.
We also retrieved virus-host protein interactions from the
literature and added them to the VirHostNet knowledge base
(37). In total, 26 interactions were identified involving 23 hu-
man proteins. (All of the papers describing individual interac-
tions can be retrieved using Table S1 in the supplemental
material, where we have indicated the PubMed ID number.) In
addition, Shapira et al. identified recently 90 interactions in-
volving 69 cellular proteins in their Y2H screens with the viral
polymerase subunits (51). Moreover, we added 49 cellular pro-
teins that were identified as vRNP or polymerase complex host
cell partners by proteomic approaches, even though we don’t
know precisely with which viral protein they actually interact
(23, 33). The overlap between these four data sets is low, but
not surprising (Table 2). (Note that these data sets include the
Y2H set from this report, which is further referred to as the
“I-MAP” [for “Infection Mapping Project”] data set, the Y2H
set from reference 51, and the literature curated [LC] and
proteomics [affinity purification; AP] sets.) In a previous work,
where we performed a comprehensive analysis of the hepatitis
C virus (HCV) interaction network, we obtained such results:
among 278 human proteins interacting with HCV proteins
identified in the Y2H set, only 10 were already described in the
literature, representing 3.6% overlap (9). This is due to the
different methods that were used (e.g., the Y2H assay for
I-MAP versus affinity purification-mass spectometry [MS] for
the AP set, representing 1.8% overlap). The low overlap
(0.9%) between our results and those from Shapira et al. (51),
although both studies were conducted using the Y2H assay,
could be explained by different procedures that were used,
including the following: different libraries (a normalized library
of 12,000 full-length ORFs for the study by Shapira et al. versus
3 human cDNA libraries encompassing a large fraction of the
human proteome, but including many truncated coding se-
quences for I-MAP), different screening techniques (mating of
a minipool of prey strains for the study by Shapira et al. versus
mating of one bait strain against a whole library of prey strains
for I-MAP), different Y2H strains (Y8800 and Y8930 for the
study by Shapira et al. versus AH109 and Y187 for I-MAP),
and different reporter genes (ADE2 and HIS3 for the study by
Shapira et al. versus HIS3 for I-MAP) (51). This is supported
by a comprehensive interactome analysis conducted by the
Vidal lab (29), in which Y2H screens were conducted in par-
allel against both cDNA and ORFeome libraries and then
compared for the protein interactions identified. In total, 1,517
interactions were identified with the cDNA library and 3,263
with the ORFeome library. Only 250 interactions were discov-
ered using both libraries, thus representing 5.5% overlap. Fur-
thermore, the viral polymerase genes used as baits came from
different influenza virus strains compared to the results from
Shapira et al. (51) and our I-MAP data set.
The FluPol interactome we reconstructed is thus composed
of 4 viral proteins and 234 human partners (HFluproteins)
forming 279 viral-human protein interactions (Fig. 1). For a
dynamic version of the network, see http://flupol.lyon.inserm.fr
and Table S1 in the supplemental material). Using VirHostNet
(37), we found that among the 234 HFluproteins, 111 are
connected with each other through 204 interactions within the
human interactome (HFlu-HFlu) (Fig. 1; see Table S2 in the
supplemental material). We also found through VirHostNet
that 69 HFluproteins are targeted by 47 other viruses (see
Table S3 in the supplemental material). This suggests that
cellular pathways that are connected to the influenza virus
polymerase (see below) represent common virus targets. Fur-
thermore, 24 influenza virus polymerase interactors are also
connected to other influenza A virus proteins (mainly NS1; see
Table S4 in the supplemental material), centering the polymer-
ase partners in a more comprehensive influenza virus network.
Functional enrichment of the influenza virus polymerase
interactome. To visualize which cellular functions are targeted
by the viral polymerase, the enrichment of HFlufor Gene
Ontology (GO) terms corresponding to “biological processes”
was determined by using the BiNGO plugin in Cytoscape (1,
16, 32, 50). It is biased, since not all human proteins have yet
been annotated (in our data set, 208 of the 234 HFluproteins
possess a GO “biological process” annotation), but it remains
a powerful way to incorporate conventional biology to system-
level data sets. We took into account the false-positive rate
obtained during the validation of the Y2H interactors and
performed 50 tests by removing randomly 15% of the Y2H
cellular partners. We retained only GO terms that were signif-
icantly enriched in all 50 tests. Functions related to DNA
synthesis (DNA replication and DNA repair), transcription,
RNA processing, and translation were overrepresented. An
enrichment for cell cycle, nucleocytoplasmic transport, re-
sponse to unfolded proteins, and viral infectious cycle was also
found (Fig. 1C). Among these overrepresented processes, sev-
eral were mainly retrieved from literature—e.g., nucleocyto-
plasmic transport (10/14 cellular interactors were mined from
literature) or DNA replication (6/10 interactors from litera-
ture)—denoting functions already known to be associated with
the influenza virus polymerase (11, 13, 25, 34, 35, 39, 46, 57).
Other processes contain several interactors discovered in our
Y2H screens, like RNA processing (11/27 proteins identified
TABLE 2. Overlap in FluPol interactors between different data sets
No. of HFlu
No. (%) of interactors with overlapa:
et al. (51)
et al. (51)
1 (0.9)3 (2.8)
0 1 (1.4)
aThe overlap is represented as percentage of the total HFludata set analyzed
(horizontally). For example, the overlap of 1 interactor between Y2H I-MAP and
Y2H Shapira et al. corresponds to 0.9% of the 109 HFluproteins identified in
VOL. 85, 2011HOST CELL PARTNERS OF THE INFLUENZA VIRUS POLYMERASE13013
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FIG. 1. The influenza virus polymerase (FluPol) interaction network. (A) Graphical representation of the FluPol interactome map. Black nodes
represent viral proteins. V represents NP, PA, PB1, and PB2. Pol corresponds to the polymerase complex, and RNP to the ribonucleoprotein particle,
composed of all 4 proteins and vRNA. Gray nodes represent human proteins (HFlu). Red, orange, and green edges represent interactions between viral
and human proteins: V-HFlu, identified by the Y2H assay (I-MAP data set) is in red, literature mining is in orange, and affinity purification is in green.
Black edges represent interactions between viral proteins (V-V), and blue edges represent interactions between human proteins (HFlu-HFlu). The FluPol
interaction network is available online; for a better visualization and dynamic display, see http://flupol.lyon.inserm.fr. Human proteins of enriched classes
are colored according to the table in panel C. (B) Number of FluPol interactors. Data are given for the Y2H data set, literature curated interactions (LC),
and the affinity purification (AP) approach by viral protein, the polymerase, and the vRNP (for the AP approaches). (C) Table of the overrepresented
indicated are the number of targeted proteins (which are detailed in the HFlucolumn) and the total proteins within each class. A color was assigned to
each overrepresented class, allowing their positioning on the interactome map.
on November 21, 2011 by guest
from Y2H assay), DNA repair (8/18 proteins identified from
Y2H assay), unraveling physical links between the influenza
virus polymerase and these cellular functions. They may be
mandatory for the function of influenza virus polymerase
within the host cell.
Functional implication of the FluPol partners. On the basis
of the enriched cellular functions that are targeted by the
influenza virus polymerase, we selected eight validated cellular
interactors to test their ability to affect the viral replication:
C14orf166 (not annotated in GO but known as a transcription
elongation factor) (20, 44), COPS5 (implicated in transcription
and in translation), EIF3S6IP (translation), FANCG (DNA
repair), MNAT1 (a member of 4 overrepresented classes, in-
cluding cell cycle, RNA processing, transcription, and DNA
repair), NMI (transcription), NUP54 (nucleocytoplasmic
transport), and PRKRA (RNA processing and also implicated
in innate antiviral response and known to interact with NS1)
(our unpublished data and reference 39) (Fig. 1C).
To further confirm observed interactions, we tested whether
these cellular partners interact with the viral polymerase in
influenza virus-infected cells. For this purpose, we transfected
cells with each protein fused to the 3? Flag epitope and then
infected them with H1N1 A/New Caledonia/2006 virus (clinical
isolate). Twenty-four hours after infection, Flag-tagged cellu-
lar proteins were immunoprecipitated, and Western blots were
performed with an antibody raised against PB1 to detect the
presence of the polymerase in the immunoprecipitates. As
shown in Fig. 2, COPS5, EIF3S6IP, FANCG, NMI, NUP54,
and PRKRA immunoprecipitated the viral polymerase (detec-
tion of PB1 in the immunoprecipitates). We did not detect PB1
in the C14orf166 and MNAT1 immunoprecipitates. Neverthe-
less, we noticed that C14orf166 interacts with PA but not with
the other polymerase subunits (Table 1). This might reflect
that C14orf166 only interacts with free PA protein. In sum-
mary, these results indicate that 6 of the 8 host cell factors
tested specifically associate with the RdRP in an infected cell
We then monitored the virus replication in human cells
where these 8 influenza virus polymerase interactors were de-
pleted by specific siRNA. We added to this set POLR2A,
FIG. 2. Interaction of the influenza virus polymerase and host cell
partners in infected cells. A549 cells were transfected with the indi-
cated proteins fused to 3? Flag. Twenty-four hours after transfection,
cells were infected with H1N1 A/New Caledonia/2006 at an MOI of 1.
Cells were lysed 24 h after infection, and protein lysates were immu-
noprecipitated (IP) with anti-Flag (?-Flag) magnetic beads. Total cell
extracts and immunoprecipitates were separated by SDS-PAGE and
analyzed by Western blotting using antibodies against PB1 (?-PB1)
FIG. 3. FluPol-host cell interactors regulate virus replication. (A) Schematic representation of the replication assay. Human A549 cells treated
with targeted siRNA were infected 48 h after depletion by H1N1 A/New Caledonia/2006 at an MOI of 0.5. Cell culture supernatants were harvested
48 h after infection, and viral titers were determined by a MUNANA assay (measuring the neuraminidase [NA] activity). (B) Results (mean of
2 independent experiments, each performed in triplicate) are expressed as relative replication efficiency compared to cells treated with a scrambled
siRNA control. (C) To assess potential cytotoxicity induced by the siRNAs, a cell viability assay was performed using the resazurin-based
fluorometric assay 48 h after siRNA transfection. Cells were counted and expressed as relative to control cells (treated with a scrambled siRNA
VOL. 85, 2011HOST CELL PARTNERS OF THE INFLUENZA VIRUS POLYMERASE 13015
on November 21, 2011 by guest
which we identified by Y2H assay and which was previously
described in the literature as an RdRP interactor (14). For this
purpose, siRNA-depleted cells were infected by H1N1 virus,
and 48 h after infection, virus titers were determined by
quantifying the neuraminidase activity using a MUNANA
assay (59) (see Materials and Methods). The individual de-
pletion of 8 genes decreased the virus replication to less
than 50% compared to a control scrambled siRNA:
C14orf166, COPS5, EIF3S6IP, FANCG, MNAT1, NMI,
NUP54, and POLR2A (Fig. 3B). Conversely, PRKRA ap-
peared to have potent antiviral activity, as its inhibition by
siRNA increased the virus replication 3.5 times compared to
the control scrambled siRNA. As a control, we showed that
tested siRNA had no side effects on cell survival as assessed
by a resazurin assay (Fig. 3C).
We speculated that these cellular proteins specifically regu-
late the viral polymerase activity. This was tested in a minige-
nome replicon assay either by overexpressing (by transient
transfection of a plasmid) or by depleting (using directed
siRNA) each cellular partner gene. HEK-293T cells were
cotransfected with vectors encoding the A/WSN/33 polymerase
subunits and NP and a reporter plasmid encoding an RNA
template for the viral polymerase that expresses the firefly
luciferase, together with a plasmid expressing each cellular
partner. Luciferase activity was determined 48 h after trans-
fection. For the depletion assay, human cells were first treated
by siRNA targeting each gene for 2 days and then were trans-
fected with the complete set of the vRNP (plasmids expressing
NP, PA, PB1, and PB2 and the reporter plasmid). Results are
presented in Fig. 4. The eight genes required for virus repli-
cation reveal an increased polymerase activity when overex-
pressed and/or a decreased activity when depleted. COPS5,
EIF3S6IP, FANCG, and MNAT1 overexpression enhanced
the RdRP transcriptional activity from 1.25 to 3 times com-
pared to the control. The depletion of C14orf166, COPS5,
EIF3S6IP, NUP54, and POLR2A reduced more than 2 times
the viral polymerase transcription. We did not observe a dras-
tic change in the RdRP transcription when overexpressing or
depleting NMI. This cellular protein could contribute to viral
replication at specific steps that cannot be detected with the
minigenome system, like virus assembly or trafficking (2, 62).
These human proteins are thus host cell factors required for
influenza virus polymerase activity, a mandatory step for influ-
enza virus replication. On the contrary, PRKRA acts as an
antiviral cellular factor, since its overexpression inhibits the
viral polymerase activity and its knockdown favors the influ-
FIG. 4. FluPol-host cell interactors regulate the viral polymerase activity. (A) Schematic representation of the viral polymerase transcriptional
activity using a minigenome replicon assay. Human 293T cells were either transfected by a vector encoding the cellular interactor (section 1,
overexpression test) or treated with a siRNA targeting the cellular gene (section 2, depletion test). For the overexpression assay (in blue), 293T
cells were transfected with a plasmid encoding each cellular protein together with NP, PA, PB1, PB2, a minigenome replicon reporter plasmid
coding for firefly luciferase, and a Renilla control vector. Forty-eight hours posttransfection, the luciferase activities were determined. For the
depletion assay (in red), 293T cells were first treated with specific siRNAs targeting cellular partners’ genes. Forty-eight hours after depletion, cells
were transfected with the complete set of vRNP (NP, PA, PB1, and PB2 and the luciferase reporter plasmid) and the Renilla control vector, and
the experiment proceeded as for the overexpression test. (B) Results (mean of 2 independent experiments, each performed in triplicate) are
expressed as relative effect of overexpression (in blue) or knockdown of FluPol interactors (in red) compared to the control (empty vector for
overexpression or scrambled siRNA for depletion). As a negative control (ctl ?), cells were transfected with the same reaction mixture without
PB1.*and**, P ? 0.05 and P ? 0.01, respectively, compared to the positive control (ctl ?), based on a Student’s t test.
13016 TAFFOREAU ET AL.J. VIROL.
on November 21, 2011 by guest
enza virus polymerase transcriptional activity as well as the
Among the eight proviral cellular proteins, 5 have been
implicated in mRNA transcription: C14orf166, COPS5,
MNAT1, NMI, and POLR2A. The latter protein, the largest
polymerase II (Pol II) subunit, interacts with the viral poly-
merase through its carboxy-terminal domain (CTD) phosphor-
ylated at serine 5 (i.e., when the transcription initiates and
where the capping enzymes are recruited and activated) (14).
This interaction inhibits the transcription elongation and leads
to the degradation of POLR2A (6, 47). We noticed in a very
recent paper that influenza virus polymerase interacts with the
P-TEFb complex (CDK9/cyclin T1), which phosphorylates the
Pol II CTD at the onset of transcription elongation (61). This
further connects the influenza virus polymerase complex to the
RNA Pol II at the beginning of the transcription elongation
(i.e., when cellular pre-mRNAs are capped) (reviewed in ref-
erence 8). We have shown that the POLR2A CTD specifically
and directly interacts with PA: the clones isolated in the Y2H
assay all correspond to the POLR2A CTD, and in the FCPI
assay, it interacted only with PA (and not with NP, PB1, or
PB2) (Table 1). In biochemical fractionation experiments, the
actively transcribing Pol II form and many proteins involved in
cellular transcription and RNA processing are associated with
an insoluble nuclear fraction, the nuclear matrix (36). Inter-
estingly, newly synthesized viral RNA and components of the
vRNP complex have been found associated with this nuclear
matrix in influenza virus-infected cells, and protein interac-
tions identified in this study probably participate in this process
PRKRA, which we identified as a protein required for the
host antiviral response, is a double-stranded RNA (dsRNA)
binding protein that activates protein kinase R (PKR) by in-
teracting through their dsRNA binding domains (41). PKR, a
key mediator of the interferon antiviral pathway, is a kinase
that phosphorylates the translation initiation factor eIF2?,
leading to protein synthesis shutoff (7). The overexpression of
PRKRA in mammalian cells leads to PKR activation and in-
hibition of translation and, under cellular stress, to apoptosis
(40, 41). It has already been shown that PRKRA interacts with
viral proteins, as the dsRNA binding protein Us11 of HSV1.
This interaction inhibits PKR activation and the PRKRA-in-
duced apoptosis (45). Recently, vesicular stomatitis virus
(VSV) replication was monitored in a PRKRA-dependent
context, showing that the virus production is enhanced in
PRKRA-depleted cells and that PRKRA overexpression pro-
tects cells against VSV infection (3), like we observed in influ-
enza virus replication assay. Furthermore, it has also been
shown that the influenza virus NS1 protein interacts with both
PRKRA and PKR, leading to the inhibition of PKR and stress-
induced cell death mediated by PRKRA (30). It appears thus
that the PRKRA-PKR pathway is very specifically targeted by
the influenza virus, and this might be an important way for the
virus to escape the innate immune response in infected cells.
In their comprehensive influenza virus infection regulatory
study, Shapira and colleagues identified the viral polymerase
subunits as modulating factors of the host antiviral response,
like the interferon production pathway (51). Furthermore, in-
fluenza virus RdRP was recently shown to interact with Stau1,
a dsRNA binding protein and well-characterized target of NS1
This global approach paves the way to a new landscape of
the influenza virus polymerase cellular neighborhood. The in-
fluenza virus polymerase interaction network will help us to
better decipher the virus biology and molecular mechanisms of
viral replication. By adding a functional correlation between
the influenza virus polymerase and cellular cofactors, it will
facilitate the development of new antiviral drugs that target the
host cell factors instead of viral proteins, the latter being more
prone to mutate and become drug resistant.
We thank D. Hart (H3N2 A/Hong Kong/43/75 PB2 ORF), G.
Brownlee (H1N1 A/WSN/33 ORFs encoding vectors), V. Moule `s
(H1N1 A/PR/8/34 genomic cDNA, H5N1 A/VietNam/1194/2004 and
H5N1 A/Turkey/651242/2006 genomic RNA), J. Ortin (polyclonal an-
tibody against PB1), Y. Jacob (pCMV-eGFP-gw and pCI-neo-
3Flag-gw vectors), and M. Shaw (minigenome reporter plasmid) for
providing materials. We also thank all of the members of the I-MAP
team for fruitful discussions.
This work was supported by the EU FLUPOL (SP5B-CT-2007-
044263), ANR FLU INTERPOL (ANR-06-MIME-014-02), the
French Fonds Unique Interministériel (FUI), and INSERM.
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