JOURNAL OF VIROLOGY, Feb. 2010, p. 1254–1264
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 3
Nuclear Import and Assembly of Influenza A Virus RNA
Polymerase Studied in Live Cells by Fluorescence
Se ´bastien Huet,1Sergiy V. Avilov,1,2,3Lars Ferbitz,2‡ Nathalie Daigle,1
Stephen Cusack,2* and Jan Ellenberg1*
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany1;
Grenoble Outstation, European Molecular Biology Laboratory, 6 rue Jules Horowitz, 38042 Grenoble cedex 9, France2; and
Palladin Institute of Biochemistry, 9 Leontovich str., Kiev 01030, Ukraine3
Received 23 July 2009/Accepted 4 November 2009
Intracellular transport and assembly of the subunits of the heterotrimeric RNA-dependent RNA polymerase
constitute a key component of the replication cycle of influenza virus. Recent results suggest that efficient
polymerase assembly is a limiting factor in the viability of reassortant viruses. The mechanism of nuclear
import and assembly of the three polymerase subunits, PB1, PB2, and PA, is still controversial, yet it is clearly
of great significance in understanding the emergence of new strains with pandemic potential. In this study, we
systematically investigated the interactions between the polymerase subunits and their localization in living
cells by fluorescence cross-correlation spectroscopy (FCCS) and quantitative confocal microscopy. We could
show that PB1 and PA form a dimer in the cytoplasm, which is imported into the nucleus separately from PB2.
Once in the nucleus, the PB1/PA dimer associates with PB2 to form the trimeric polymerase. Photon-counting
histogram analysis revealed that trimeric polymerase complexes can form higher-order oligomers in the
nucleus. We furthermore demonstrate that impairing the nuclear import of PB2 by mutating its nuclear
localization signal leads to abnormal formation of the trimeric polymerase in the cytoplasm. Taken together,
our results demonstrate which of the previously discussed influenza virus polymerase transport models
operates in live cells. Our study sheds light on the interplay between the nuclear import of the subunits and
the assembly of the influenza virus polymerase and provides a methodological framework to analyze the effects
of different host range mutations in the future.
Influenza A viruses can infect a wide range of avian and
mammalian species (49). Most avian strains of influenza virus
infect wild waterfowl and domestic poultry but usually do not
spread to humans. However, adaptation of pathogenic avian
viruses to humans can occur either by mutation or reassort-
ment, leading to potentially very serious pandemics, as was the
case in 1918 when the “Spanish” flu caused 20 to 40 million
deaths worldwide (33). Due to this ability to cross the species
barrier, influenza A viruses are a permanent threat to human
health. Since 2005 the spread of highly pathogenic H5N1 avian
strains in Asia, Europe, and Africa has raised serious concern
about the potential of this strain to cause an influenza pan-
demic (50). Since early 2009, an ongoing new, rapidly evolving
pandemic threat has arisen from the emergence of a highly
contagious, interhuman-transmissible “quadruple reassortant”
swine H1N1 virus to which the world population is antigeni-
cally naïve (6).
Influenza A viruses are enveloped viruses of the ortho-
myxovirus family whose genomes comprise eight negative-
strand RNA segments (2). In contrast to many RNA viruses,
the influenza virus genome is transcribed and replicated by
the trimeric viral RNA polymerase (PA, PB1, and PB2)
in the nuclei of the infected cells. Therefore, the polymerase
subunits, which are produced in the cytoplasm, have to be
imported into the nucleus and assembled into a functional
trimer (2, 18). Many studies have demonstrated that the
viral polymerase plays a major role in host specificity, prob-
ably due to the necessity for the polymerase subunits to
adapt to host cell-interacting partners such as nuclear im-
port factors (13, 16, 25, 37, 46). Due to the lack of in vivo
data concerning the interactions between the polymerase
subunits in the nucleus and the cytoplasm of the host cells,
the mechanisms of polymerase assembly and nuclear import,
as well as their spatial and temporal relationships, are still
not completely understood. Putative nuclear localization
signals (NLSs) have been identified on PB1 (31), PB2 (29),
and PA (32), suggesting that each subunit could be imported
separately. However, based on in vitro assembly observa-
tions and cellular localization studies (8, 9, 12), it has been
proposed that PB1 and PA are imported into the nucleus as
a subcomplex by import factor RanBP5 (a member of the
importin ? superfamily). PB2 is thought to enter the nucleus
separately, probably via the canonical importin ?/importin ?
* Corresponding author. Mailing address for Jan Ellenberg: Cell
Biology and Biophysics Unit, European Molecular Biology Labora-
tory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Phone: 49
6221 387-8328. Fax: 49 6221 387-8518. E-mail: email@example.com.
Mailing address for Stephen Cusack: Grenoble Outstation, European
Molecular Biology Laboratory, 6 rue Jules Horowitz, 38042 Grenoble
cedex 9, France. Phone: 33 47620-7238. Fax: 33 47620-7786. E-mail:
† Supplemental material for this article may be found at http://jvi
‡ Present address: Lonza Ltd., CH-3930, Visp, Switzerland.
?Published ahead of print on 11 November 2009.
pathway (46), and then associates with the PB1/PA het-
erodimer in the nucleus to form the functional trimeric
polymerase. Nevertheless, alternative pathways have also
been proposed. Naito et al. (30) suggested that the nuclear
import of PB1 requires the formation of a PB2/PB1 het-
erodimer, stabilized by Hsp90, in the cytoplasm, while PA is
transported in the nucleus separately. More recently, a path-
way in which the PA/PB2 heterodimer would be formed in
the cytoplasm and then imported into the nucleus has been
proposed (17). It has also been recently shown that efficient
assembly of the trimeric polymerase could be a major lim-
iting factor in the viability of reassortant influenza viruses
(26). Since gene reassortment is an evolutionary mechanism
of influenza virus which can lead to new strains with pan-
demic potential, a precise understanding of the processes
leading to the formation of an active viral polymerase in the
nuclei of infected cells is of great importance.
Recent publications have demonstrated that fluorescence
cross-correlation spectroscopy (FCCS) is a method of choice to
study protein-protein interactions in vivo (23, 27, 42). FCCS is
the dual-color extension of fluorescence correlation spectros-
copy (FCS), a technique based on the analysis of the temporal
fluorescence fluctuations arising from single fluorescently la-
beled molecules diffusing in and out of the femtoliter-scale
detection volume commonly obtained with a confocal micro-
scope. From the autocorrelation of the fluctuating signal, it is
possible to extract the local concentrations and mobilities of
the molecules of interest (10, 28, 39). In the case of FCCS,
signals from two spectrally separated dyes labeling two differ-
ent molecules are recorded. If the two molecules interact with
each other, they diffuse synchronously through the detection
volume, resulting in correlated fluctuations in the fluorescence
signals acquired in the two channels. The cross-correlation
between the two signals is then a direct and quantitative read-
out of the interactions between the molecular species studied
(22, 38, 40). To our knowledge, this study is the first application
of FCCS to viral protein interactions and thus provides a gen-
eral methodological framework to analyze the effects of differ-
ent host range mutations and the interactions of viral proteins
and host factors in the future.
In this study, we applied FCCS to monitor the interactions
between the subunits of influenza A virus RNA polymerase in
live cells. Based both on the study of these interactions in the
cytoplasm and nucleus and on the quantitative analysis of the
intracellular localization of the subunits, we show that PB1 and
PA form a heterodimer in the cytoplasm while PB2 remains a
monomer in this compartment. Association of PB1/PA with
PB2 to form the trimeric polymerase was detected only in the
nucleus, arguing that the PB1/PA heterodimer is normally im-
ported separately from PB2. Interestingly, when we impaired
the nuclear import of PB2 by mutating its nuclear localization
signal, this induced the aberrant presence of the trimeric poly-
merase in the cytoplasm and led to the retention of PB1 and
PA outside the nucleus. Finally, by comparing the molecular
brightnesses of the single polymerase subunits with that of the
trimeric complex, we show that trimeric polymerase complexes
can interact with each other in the nucleus to form higher-
MATERIALS AND METHODS
Fluorescent protein constructs. The Clontech Living Color plasmid backbone
(Clontech, Mountain View, CA) was used to drive expression of fluorescently
labeled PB1, PB2, and PA from influenza virus strain A/Victoria/3/75(H3N2) (7)
in mammalian cells. For each construct, the entire coding sequence of the
polymerase subunit was fused to the entire coding sequence of monomeric
enhanced green fluorescent protein (mEGFP) (44), monomeric cyan fluorescent
protein (mCFP) (a kind gift of J. Lippincott-Schwartz, National Institutes of
Health, Bethesda, MD), or mCherry (41). It has been shown previously that GFP
tags do not inhibit the transcriptional and replicative activities of the polymerase,
except when fused at the N terminus of PB1 or PA (12). We therefore used PB1
and PA subunits tagged at the C terminus, and PB2 was tagged at its N terminus.
pmEGFP-PB2 and pmEGFP-PB2-QNQ (a PB2 mutant with three point muta-
tions [K738Q, K752N, and R755Q] at its C terminus [see Results]) have been
described previously (46). In mCFP-PB2 and mCherry-PB2, an SGLRSRAQ
linker connects the two fused proteins. In PB1-mEGFP, PB1-mCFP, and PB1-
mCherry, the linker is AAAVPRARDPPVAT, and in PA-mEGFP, PA-mCFP,
and PA-mCherry, it is TRDPPVAT.
Cell culture and transfection. HeLa Kyoto and A549 cells were routinely
cultured in phenol-red free Dulbecco’s modified Eagle’s medium (4.5 g/liter
glucose) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 ?g/ml
penicillin, 100 U/ml streptomycin, and 1 mM sodium pyruvate in 5% CO2at
37°C. At 48 h prior to experiments, cells were plated on LabTekII chambered
cover glasses (Nunc, Roskilde, Denmark), and then they were transfected using
Fugene 6 (Roche, Basel, Switzerland) or jetPRIME (Polyplus Transfection,
Illkirch, France) 24 h later. For imaging and FCCS acquisitions, growth medium
was replaced by a CO2-independent medium without phenol red (Invitrogen,
Carlsbad, CA) supplemented with 20% fetal bovine serum, 2 mM glutamine, 100
?g/ml penicillin, and 100 U/ml streptomycin. All the experiments were per-
formed at 33°C.
Imaging and FCS/FCCS setup. Imaging and FCS/FCCS experiments were
performed on a TCS SP2 acousto-optic beam splitter (AOBS) confocal micro-
scope (Leica Microsystems, Wetzlar, Germany) using a water immersion Apo-
chromat 63? objective lens (numerical aperture [NA], 1.2). The monomeric cyan
fluorescent protein (mCFP) and the monomeric enhanced green fluorescent
protein (mEGFP) were excited with the 458-nm and 488-nm lines of an argon
laser, respectively. mCherry was excited using a 561-nm diode laser. For the
FCS/FCCS acquisitions, the laser power at the sample level was set to approxi-
mately 1 ?W for the different laser lines in order to minimize photobleaching.
The size of the detection pinhole was set to 1 Airy unit. For imaging, fluorescence
signals were detected using photomultiplier tubes. To separate the light emitted
by the different fluorophores, the following spectral windows were chosen for the
AOBS: 460 to 500 nm for mCFP, 500 to 550 nm for mEGFP, and 590 to 670 nm
for mCherry. To minimize cross talk between mCFP and mEGFP, cells were first
imaged in the cyan channel and then imaged simultaneously in the green and red
channels. FCS/FCCS raw data were acquired using a specific detection module
consisting of two avalanche photodiodes (APDs) (Perkin-Elmer Optoelectronics,
Fremont, CA) coupled to a photon-counting card (ISS, Champaign, IL) allowing
the registration of the raw intensity data at 24 MHz in the photon delay mode.
Ahead of the APDs, fluorescence light was split using a dichroic mirror (LP560)
and filtered spectrally by passing through band-pass emission filters (BP500-550
and HQ638DF75; Omega Optical, Brattleboro, VT). For each FCS/FCCS mea-
surement, 2 ? 105to 5 ? 105photons were typically acquired.
FCS/FCCS data analysis. (i) Fitting of the FCS/FCCS curves. Auto/cross-
correlation curves were calculated from raw intensity data using the FFS Data-
processor 2.0 software (Scientific Software Technology Center, Minsk, Belarus).
For time delays ? from 5 ?s to 1 s, the auto/cross-correlation curves were fitted
with a one-species model including the contribution of the fluorophore photo-
physics and assuming that the intracellular diffusion of the labeled subunits is
anomalous (see the supplemental material for more details). From these fits of
the autocorrelation curves, we could estimate the parameter Neff, which refers to
the mean number of fluorescent particles within the detection volume. For the
cross-correlation curves, Neff? NiNj/Nij, where Ni, Nj,and Nijare the mean
numbers of particles within the detection volume detected in channel i, in
channel j, and in both channels, respectively. As the size of the detection volume
was equal to 0.7 to 1 fl (see the supplemental material), it was then possible to
estimate the concentration of the labeled subunits in the probed cells.
The fits of the auto/cross-correlation curves also give access to the parameter
?diff, which corresponds to the characteristic diffusion time of the fluorescent
molecules within the detection volume. ?diffdepends both on the diffusion coef-
ficient of the protein and on the size of the detection volume (40). Consequently,
to analyze the diffusion properties of the influenza virus polymerase subunits, we
VOL. 84, 2010ASSEMBLY OF INFLUENZA VIRUS POLYMERASE PROBED BY FCCS1255
instead used the relative diffusion time given by ?diff,rel? ?diff/?diff,mCherry, where
?diff,mCherryis the diffusion time measured for uncoupled mCherry in the same
compartment, which serves to calibrate the detection volume.
(ii) Determination of the proportion of the viral polymerase subunits engaged
in a complex. FCCS was performed between the mEGFP- and the mCherry-
labeled subunits. With our current setup, it was not possible to cross-correlate
fluorescence signals from more than two channels. Consequently, when study-
ing the formation of the trimeric polymerase complex, only the interactions
between the mEGFP- and the mCherry-labeled subunits could be monitored by
FCCS. The third subunit was labeled with mCFP to control its expression in the
analyzed cell and to quantify its intracellular concentration (see the supplemen-
tal material for more details). In order to characterize the formation of dimeric
or trimeric complexes between the polymerase subunits, we quantified the pro-
portion of the least concentrated subunit engaged in a complex. For all these
FCCS experiments, the least concentrated subunit was the mCherry-labeled one.
The calculated proportions of subunit engaged in a complex were corrected for
the nonperfect overlap between the detection volumes in the two channels and
for the bleed-through from mEGFP into the red channel (see the supplemental
material for more details).
Measurements of molecular brightness by photon-counting histogram analy-
sis. The molecular brightness of the mEGFP-labeled polymerase subunits was
estimated by photon-counting histogram (PCH) analysis. Raw intensity data
were acquired using the same experimental settings as the ones used for FCS/
FCCS acquisitions. Histograms of the numbers of photons per time bin of 100 ?s
were calculated from the raw intensity data using the FFS Dataprocessor 2.0
software. The photon histograms were fitted with a one-species model assuming
a three-dimensional Gaussian detection volume (see the supplemental material
for more details). From the analysis of the PCH curves, we estimated the mean
number of fluorescent particles within the detection volume Neff
tive molecular brightness of these particles εeff(in counts per second per mole-
cule). As the time binning used to calculate the photon histograms was in the
same range as the characteristic time spent by the fluorescent molecules within
the detection volume, the values obtained for Neff
for the diffusion of these molecules using the approach described by Perroud et
al. (34). For the cells coexpressing the three fluorescently tagged polymerase
subunits, we controlled by FCS that the mEGFP labeled subunit was the least
concentrated one (see above).
Statistical analysis. For each quantitative analysis described in this study, the
measurements were performed on 8 to 14 different cells per condition (for the
FCCS experiments, this also includes measurements performed on positive and
negative control cells [see the supplemental material for more details]). The
values presented correspond to an average of these measurements and are given
as mean ? standard deviations (SD). The significance of differences was calcu-
lated using Student’s unpaired t test.
PCHand the effec-
PCHand εeffhad to be corrected
Influenza virus polymerase subunits influence each other’s
subcellular localization. Fluorescently labeled PB1, PA, and
PB2 viral polymerase subunits were first expressed separately
to study their intracellular localization. Strikingly, each of the
three subunits showed a very different subcellular pattern. PB2
was localized nearly exclusively in the nucleus, while PA was
present at similar concentrations in the cytoplasm and in the
nucleus and PB1 was strongly accumulated in the cytoplasm
(Fig. 1 and Table 1). While similar localization has been re-
ported for PB2 and PA (12, 19, 46), the cytoplasmic localiza-
tion of PB1 is in contrast with previous studies showing a
nuclear or uniform distribution (30, 31, 43), which may be due
to the use of cell lines from different organisms.
Next, we analyzed the localization of each polymerase sub-
unit in the presence of its binding partners in all heterodimeric
and trimeric combinations and quantitated the effect on the
localization by measuring the nuclear/cytoplasmic concentra-
tion ratio (Fig. 1 and Table 1). In order to study how the
localization of a given subunit is affected by the presence of
another subunit(s) quantitatively, it was necessary to ensure
that these subunits were in excess over the subunit whose
localization was analyzed. We therefore measured the relative
concentrations of all the subunits expressed in the cytoplasm of
transfected cells by FCS, and the intracellular distribution of
the subunit with the lowest cytoplasmic concentration was then
monitored by quantitative confocal imaging. The localization
pattern of PB1 and PA in the case of their coexpression was
first characterized. Published reports suggest that these two
subunits could be imported efficiently into the nucleus as a
heterodimer (9, 12). Our experiments showed that when ex-
pressed together, PB1 and PA tended to relocalize into the
nucleus, compared to the cells expressing only PA or PB1 (Fig.
1 and Table 1). The efficiency of the nuclear accumulation of
these subunits was, however, quite low, as the nucleus/cyto-
plasm concentration ratios were lower than 2 for both PB1 and
PA. Further, we studied how PB2 affects the intracellular dis-
tribution of either PB1 or PA. PB2 had no effect on the local-
ization of PA or PB1.
Finally, the three polymerase subunits were coexpressed si-
multaneously. In the presence of their two binding partners,
both PB1 and PA were localized nearly exclusively in the nu-
cleus (Fig. 1 and Table 1). In order to control that the subunit
localizations observed in HeLa cells were not specific to this
cell line, we also analyzed these localizations in A549 cells (see
Fig. S1 in the supplemental material). The results we obtained
with A549 cells were similar to those with HeLa cells.
The observation that the nuclear accumulation of PB1 and
PA is more efficient when both subunits are coexpressed in the
presence of PB2 can be interpreted in two different ways: (i)
the trimeric polymerase could be formed in the cytoplasm and
then imported into the nucleus more efficiently than a PA/PB1
dimer, or (ii) PB1 and PA could be imported into the nucleus
as a dimer and then interact with PB2 to form the trimeric
polymerase; this association with PB2 would lead to the reten-
tion of PA and PB1 in the nucleus. In order to determine which
of these models properly describes the mechanism occurring in
vivo, we used fluorescence cross-correlation spectroscopy to
visualize the interactions between the influenza virus polymer-
ase subunits directly.
Interactions between the influenza virus polymerase sub-
units measured by fluorescence cross-correlation spectros-
copy. In order to validate the FCCS method for measuring
protein-protein interactions in live cells, we first performed
FCCS measurement in cells expressing a construct for which
mEGFP was physically linked to mCherry through a neutral
spacer, the maltose-binding protein (positive control), and in
cells coexpressing mEGFP and mCherry (negative control).
For the positive controls we obtained strong cross-correlation
signals both in the nucleus and in the cytoplasm (Fig. 2A, left).
The amplitude of the cross-correlation curves was, however,
about half of that of the auto-correlation curves in the green
and red channels, revealing the imperfect overlap between the
two excitation volumes. The subsequent measurements of the
interactions between the influenza virus polymerase subunits
were corrected for this artifact (see the supplemental mate-
rial). For the negative controls, the amplitude of the cross-
correlation curves was close to zero (Fig. 2A, middle). The very
weak cross-correlation signal observed in these cells is due to
the bleed-through from the mEGFP fluorescence into the red
channel. This artifact was corrected when quantifying the in-
1256HUET ET AL.J. VIROL.
FIG. 1. Subcellular localization of the influenza virus polymerase subunits. (A) Subcellular localizations of PA-mEGFP (left column),
PB1-mEGFP (center column), and mEGFP-PB2 (right column) when expressed alone or in association with other subunits in HeLa Kyoto
cells. The legend at the bottom right of each image refers to the different subunits expressed by the cells. When two subunits were present,
the subunit not shown was labeled with mCherry. In cells coexpressing the three subunits, the subunits not shown were labeled with mCherry
and mCFP. This allowed to control by FCS that the shown subunit was the least concentrated in the cytoplasm. The autofluorescence was
removed from the images by spectral linear unmixing (52). Scale bar, 10 ?m. (B) Concentration ratio between the nucleus and the cytoplasm
quantified from the confocal images for PA (left), PB1 (center), and PB2 (right) in cells expressing different combinations of subunits. For
cells expressing multiple subunits, the subunit whose nucleus/cytoplasm concentration ratio was estimated was the least concentrated in the
cytoplasm as assessed by FCS.***, P ? 0.001.
VOL. 84, 2010 ASSEMBLY OF INFLUENZA VIRUS POLYMERASE PROBED BY FCCS 1257
teractions between the influenza virus polymerase subunits
(see the supplemental material).
We then studied by FCCS the interactions between the dif-
ferent polymerase subunits in the cytoplasm and the nuclei of
live HeLa cells (see Fig. 2A, right, for an example of the
experimental auto/cross-correlation curves and Fig. 2B and C
for the quantitative results). For these experiments, the intra-
cellular concentrations of the different subunits were in the
range of 10 to 100 nM, as shown by the analysis of the auto-
correlation curves (see Materials and Methods). First, FCCS
experiments were performed with cells coexpressing PB1 and
PA to study the interactions between these two subunits. We
found that almost all the PA subunit (which was the least
concentrated subunit in these experiments) was in complex
with PB1, with no significant difference (P ? 0.31) between
cytoplasm (87% ? 16% of PA in complex) and nucleus
(93% ? 10% of PA in complex). In contrast, no significant
interaction was observed between PA and PB2 in either the
nucleus or the cytoplasm. In cells coexpressing PB2 and PB1,
we observed that 28% ? 13% and 10% ? 14% (P ? 0.002) of
the least concentrated subunit (PB2 subunit for the measure-
ments in the cytoplasm and PB1 subunit for the measurements
in the nucleus) were engaged in a complex with its counterpart
in the cytoplasm and in the nucleus, respectively. Finally,
FCCS experiments were performed with cells coexpressing the
three polymerase subunits (Fig. 2B and C). Since when ex-
pressed together, PB1 and PA form a heterodimer both in the
cytoplasm and in the nucleus, we studied by two-color FCCS
the interactions between PB2 and PB1 or between PB2 and PA
in the presence of their third counterpart, assuming that PB1
and PA were in complex wherever they were coexpressed. The
two subunits whose interactions were monitored were tagged
with mEGFP and mCherry, while the third one was tagged
with mCFP to control its expression in the analyzed cell and
estimate its concentration. In the cytoplasm, we found no sig-
nificant interactions between PB2 and PA in the presence of
PB1. In contrast, 94% ? 21% of the PA subunit was in com-
plex with PB2 in the nuclei of cells coexpressing the three
subunits. Similar results were obtained when monitoring the
interactions between PB2 and PB1 in the presence of PA (data
not shown). These results show that while PB2 does not asso-
ciate with the PA/PB1 dimer in the cytoplasm, the three sub-
units interacts with each other in the nucleus to form the
trimeric polymerase. Together with the quantitative analysis of
the intracellular localizations of the subunits, these observa-
tions suggest that PB2 and the PB1/PA dimer are imported
separately into the nucleus, where they associate to form the
Impairing nuclear import of PB2 leads to the formation of
the trimeric polymerase in the cytoplasm. In order to investi-
gate the role of nuclear import of the influenza virus polymer-
ase subunits for the assembly of the trimeric complex further,
we studied the impact of impairing the nuclear import of PB2
on the interactions between the subunits and their intracellular
localizations. As reported previously (46), we observed that the
triple mutation of the C-terminal NLS domain of PB2 (K738Q,
K752N, and R755Q, hereafter referred as PB2-QNQ) strongly
reduced its nuclear accumulation (Fig. 3A and Table 1). We
then studied how PB2-QNQ affected the intracellular localiza-
tion of PA and PB1. In contrast to the case for the wild-type
PB2, coexpression with PB2-QNQ did not lead to the accumu-
lation of PA and PB1 in the nucleus (Fig. 3A and Table 1). To
assess formation of the trimeric complex, we then measured
the interactions between PB2-QNQ and PA in cells coexpress-
ing the three subunits by FCCS and found that 84% ? 17%
and 90% ? 33% of the PA subunit (which was the least
concentrated subunit in these experiments) were engaged in a
complex with PB2-QNQ in the cytoplasm and in the nucleus,
respectively (Fig. 3B and C). Control experiments performed
in the absence of PB1 showed no significant interaction be-
tween PA and PB2-QNQ in the cytoplasm (data not shown).
These results indicate that, in contrast to the wild-type PB2,
PB2-QNQ, whose nuclear import is compromised, is able to
interact with the PB1/PA dimer not only in the nucleus but also
in the cytoplasm.
The trimeric influenza virus polymerase forms higher-order
oligomers in the nucleus. Using our FCS/FCCS measurements,
we could also characterize the diffusion of the wild-type poly-
merase subunits by estimating their relative diffusion times,
?diff,rel(absolute diffusion time of the subunit divided by that of
the uncoupled mCherry [see Materials and Methods]), in the
nuclei and the cytoplasm of HeLa cells (Fig. 4). When ex-
pressed separately, PB1 and PA showed relative diffusion times
of ?15 and ?3, respectively, both in the cytoplasm and in the
nucleus. For PB2, ?diff,relwas equal to 5 ? 2 in the cytoplasm
and 11 ? 2 in the nucleus. Since in cells coexpressing PB1 and
PA the proportion of the least concentrated, mCherry-labeled,
subunit engaged in the PB1/PA dimer was close to 100%, the
diffusion time of the PB1/PA dimer can be considered to be
equal to that of the mCherry-labeled subunit. We thus find that
the PB1/PA dimer diffused approximately 15 times slower than
the uncoupled mCherry fluorophore both in the nucleus and
in the cytoplasm. Using the same approach, we estimate that in
the nucleus, the diffusion time of the trimeric polymerase was
1 order of magnitude slower than that of the mCherry control.
Based on their molecular weights and assuming that they show
a globular shape, the Stokes-Einstein hydrodynamic theory
(51) predicts that the single subunits, the PB1/PA dimer, and
the trimeric complex should diffuse ?1.6, ?2, and ?2.3 times
TABLE 1. Quantification of subcellular localizations of influenza
virus polymerase subunits
Ratio (mean ? SD) between nuclear and
cytoplasmic concn for probed subunita:
PA ? PB1
PA ? PB2
PB1 ? PB2
PA ? PB1 ? PB2
PA ? PB2-QNQ
PB1 ? PB2-QNQ
PA ? PB1 ? PB2-QNQ
0.8 ? 0.3
0.2 ? 0.1
9 ? 3
1.7 ? 0.5
0.9 ? 0.2
1.6 ? 0.5
12 ? 5
10 ? 5
9 ? 4
0.2 ? 0.1
9 ? 112 ? 6
2.6 ? 0.6
1.9 ? 0.6
0.8 ? 0.2
0.3 ? 0.4
0.8 ? 0.61.2 ? 0.6
aRatio between the nuclear and cytoplasmic concentrations quantified from
the confocal images. For cells expressing multiple subunits, the relative concen-
trations of the different subunits were first measured by FCS, and then the
localization of the least concentrated subunit in the cytoplasm was quantified.
ND, not determined.
1258 HUET ET AL.J. VIROL.
slower than the mCherry tag, respectively. The large discrep-
ancies between these predictions and the measured diffusion
characteristics of PB1, PB2, the PA/PB1 dimer, and the tri-
meric polymerase suggest that these different entities either
exist in larger complexes with host factors or form higher-order
homo-oligomers, or both.
We therefore investigated the potential formation of homo-
oligomers in the nucleus using the photon-counting histogram
(PCH) method. While FCS is based on a temporal analysis of
the intensity fluctuations arising from single fluorescently la-
beled molecules diffusing in and out the detection volume,
PCH instead characterizes the amplitude of these fluctuations.
PCH analysis gives access to the brightness and the concentra-
tion of the fluorescent diffusing particles (4). As a homo-
oligomer formed by n labeled proteins is expected to be n-fold
brighter than a single labeled protein, PCH is a powerful tool
to monitor homo-oligomerization. In order to validate the
method in the context of in vivo measurements, we first com-
pared the brightness of the uncoupled mEGFP tag with that of
an mEGFP-mEGFP tandem. As expected, the tandem con-
FIG. 2. Interactions between the influenza virus polymerase subunits studied by FCCS. (A) Examples of auto/cross-correlation curves measured
in the cytoplasm of HeLa Kyoto cells expressing mEGFP-MBP-mCherry (left), mEGFP and mCherry (center), or PB1-mEGFP and mCherry-PB2
(right). The autocorrelation curves in the mEGFP and mCherry channels are shown in green and red, respectively. The cross-correlation (CC)
curves are shown in black. The experimental curves (dotted lines) are fitted (solid lines) using a model assuming anomalous diffusion and, for the
autocorrelation curves, accounting for the photophysics of the fluorophores. (B) Proportion of the limiting subunit engaged in a complex measured
by FCCS in the cytoplasm and nuclei of HeLa Kyoto cells coexpressing pairs of subunits or the three subunits. In the double-expressing cells, the
interactions between the mEGFP- and the mCherry-labeled subunits were directly analyzed by FCCS. When the three subunits were coexpressed,
only the interaction between PA-mCherry and mEGFP-PB2 was monitored by FCCS. The presence of PB1-mCFP was assessed by imaging the
cell in the CFP channel. For the FCCS measurements of the interactions between PA and PB1 or between the three subunits, the least
concentrated subunit was PA. For the analysis of the interactions between PA and PB2, the least concentrated subunit was PB2 for the
measurements in the cytoplasm and PA for the measurements in the nucleus. For the analysis of the interactions between PB1 and PB2, the least
concentrated subunit was PB2 for the measurements in the cytoplasm and PB1 for the measurements in the nucleus. (C) Schematic description
of the interactions observed by FCCS.
VOL. 84, 2010 ASSEMBLY OF INFLUENZA VIRUS POLYMERASE PROBED BY FCCS1259
struct had nearly twice the brightness of mEGFP (Fig. 5). Next,
we quantified by PCH the brightnesses of PA-mEGFP, PB1-
mEGFP, and mEGFP-PB2 in the nuclei of cells expressing
these different subunits separately (Fig. 5). In cells expressing
PA-mEGFP or mEGFP-PB2, the brightness of the mEGFP-
labeled particles was slightly but significantly increased (P ?
0.05) compared to that of uncoupled mEGFP. This increased
brightness may indicate a small tendency of PA-mEGFP and
mEGFP-PB2 to form homo-oligomers, but we cannot exclude
that it is rather due to the coupling of mEGFP to the poly-
merase subunits, which may slightly affect the brightness prop-
erties of mEGFP. While the brightness of PA-mEGFP was not
affected by the coexpression of PB1-mCherry, expressing PA-
mEGFP together with both PB1-mCFP and mCherry-PB2
induced an increase of ?35% in the brightness of the mEGFP-
labeled particles compared to cells expressing only PA-
mEGFP (P ? 0.001). We controlled that this increase was not
due to bleed-through from the mCherry and mCFP tags into
the green channel (see the supplemental material). This ob-
servation suggests that the trimeric polymerase forms homo-
oligomers in the nuclei of HeLa Kyoto cells. The formation of
such higher-order oligomers should lead to an increase in
molecular brightness proportional to the number of poly-
merases composing these oligomers. The fact that we observed
only an ?35% increase of the molecular brightness in cells
expressing the three subunits compared to those expressing
only PA-mEGFP thus indicates that only a fraction of the
FIG. 4. Nuclear and cytoplasmic diffusions of the single influ-
enza virus polymerase subunits, PB1/PA dimers, and trimeric com-
plexes. The characteristic diffusion times of the mCherry-labeled
subunits within the detection volume were estimated by fitting the
autocorrelation curves obtained in the mCherry channel. Shown is
the ratio between these diffusion times and those measured for
uncoupled mCherry in the same compartment. These relative dif-
fusion times were measured in the cytoplasm and in the nuclei of
HeLa Kyoto cells. To characterize the diffusion of the PB1/PA
dimer, the diffusion time of PA-mCherry was estimated in cells
coexpressing PA-mCherry (least concentrated subunit) and PB1-
mEGFP. As FCCS experiments showed that most of the PA-
mCherry belongs to the PB1/PA dimer, the diffusion time of the
PB1/PA dimer was considered to be equal to that of PA-mCherry.
The same approach was used to estimate the nuclear diffusion time
of the trimeric complex.
FIG. 3. Impact of mutating the nuclear localization signal of PB2 on subcellular localization and assembly of the trimeric polymerase.
(A) Subcellular localizations of PA-mCherry (left), PB1-mCherry (center), and mEGFP-PB2-QNQ (right) in HeLa Kyoto cells coexpressing the
three subunits. The PA subunit was the least concentrated one in the cytoplasm. The autofluorescence was removed from the images by spectral
linear unmixing. Scale bar, 10 ?m. (B) Proportion of PA-mCherry subunits interacting with mEGFP-PB2-QNQ measured by FCCS in the
cytoplasm and the nuclei of HeLa Kyoto cells coexpressing PA-mCherry (least concentrated subunit), PB1-mCFP, and mEGFP-PB2-QNQ.
(C) Schematic description of the interactions observed by FCCS.
1260 HUET ET AL.J. VIROL.
polymerases are engaged in higher-order complexes. We con-
firmed the ability of the trimeric polymerase to form homo-
oligomers by monitoring the interactions between mEGFP-
PB2 and mCherry-PB2 by FCCS. While no cross-correlation
signal could be detected in the nuclei of cells expressing only
the two versions of the PB2 subunit, indicating no detectable
interaction, we observed a positive cross-correlation signal in
the presence of the other two subunits, PB1-mCFP and PA-
mCFP. For these experiments, however, it was difficult to
quantify the interactions between the trimeric polymerases as
estimated previously based on the amplitudes of the auto/
cross-correlation curves. Indeed, the different PB2 subunits
contained in an oligomer formed by several polymerases can
all be labeled with the same color. Such an oligomer will not
contribute to the cross-correlation signal, leading to an under-
estimation of the interaction. Consequently, we only estimated
the ratio between the cross-correlation curves and the auto-
correlation curves in the green channel (see Fig. S2 in the
FCCS is a powerful tool to study the interactions between
the influenza virus polymerase subunits in vivo. So far, the
cellular processes leading to the assembly of a functional in-
fluenza virus RNA polymerase inside the nucleus have been
studied mostly by in vitro biochemical techniques and imaging
of fixed cells. As these techniques could not monitor the in-
teractions between the polymerase subunits in living cells, the
role of nuclear import in the assembly of the influenza virus
polymerase has so far remained controversial. With this report,
we show that FCCS coupled with quantitative confocal imaging
and PCH analysis can be a very valuable tool to measure the
interactions between the three influenza virus polymerase sub-
units in vivo. Using this approach, we found direct evidence for
an assembly model according to which PB1 and PA het-
erodimerize in the cytoplasm and are independently imported
as a binary complex into the nucleus, where formation of the
functional PA/PB1/PB2 heterotrimer occurs through associa-
tion of PB1/PA with independently imported PB2 (Fig. 6). This
provides a direct in vivo confirmation of the model originally
proposed by Deng et al. (9).
Nuclear import and assembly of the polymerase trimeric
complex. In agreement with previous reports (12, 30), we ob-
served that the efficient accumulation of PB2 inside the nu-
cleus does not require the presence of other viral proteins. We
confirmed that the nuclear import of PB2 depends to a signif-
icant extent on its C-terminal NLS (37, 46). We also found that
the individually expressed PA and PB1 can enter the nucleus,
in agreement with previous studies (1, 12, 19), but do not
accumulate efficiently in this compartment, in contrast to PB2.
When coexpressed, PA and PB1 display a moderate nuclear
accumulation, in qualitative agreement with previous studies
(12, 32). Deng and coauthors postulated that PA and PB1 are
imported as a complex based on in vitro assembly experiments
(9). Our present FCCS experiments show that this is indeed
the case in vivo. We demonstrate that, both in the cytoplasm
and in the nucleus, almost 100% of the least concentrated
subunit (either PB1 or PA) is engaged in a PB1/PA complex. In
contrast to the coimport of PB1 and PA, we did not observe
any increase in the nuclear accumulation of PA in the presence
of PB2. In addition, FCCS experiments detected no PB2/PA
complexes, in agreement with several reports showing the lack
of direct contacts between PA and PB2 (15, 35, 47, 48). In
contrast to these results, an interaction between PB2 and PA
has recently been detected using bimolecular fluorescence
complementation (BiFC) (17). However, the biological rele-
vance of this observation remains questionable, as BiFC irre-
versibly “locks in” even very transient interactions (5). Further,
we observed that PB2 did not increase the nuclear accumula-
tion of PB1, in agreement with some previous results (12) but
in contrast to others which reported an increase of the nuclear
accumulation of PB1 in the presence of PB2 at equal or higher
FIG. 5. Homo-oligomerization of the trimeric polymerase studied
by PCH. The brightnesses of mEGFP-labeled particles were measured
by PCH in the nuclei of cells expressing uncoupled mEGFP, the
mEGFP-mEGFP tandem, or different combinations of the polymerase
subunits. In cells coexpressing PA and PB1, PA was tagged with
mEGFP and PB1 with mCherry. In cells coexpressing the three sub-
units, PA, PB1, and PB2 were labeled with mEGFP, mCFP, and
mCherry, respectively. Shown is the ratio between these brightnesses
and that of the uncoupled mEGFP.
FIG. 6. Model of the nuclear import and assembly of the influenza
virus RNA polymerase. The heterodimer formed by PA and PB1 is
imported in the nucleus separately from PB2. Once in the nucleus, PB2
binds to PB1/PA to form the trimeric polymerase.
VOL. 84, 2010 ASSEMBLY OF INFLUENZA VIRUS POLYMERASE PROBED BY FCCS1261
expression levels of the plasmid encoding PB2 (30). This dis-
crepancy may be explained by the much higher subunit con-
centrations in transfected cells used for classical fluorescence
imaging than in those suitable for FCS/FCCS experiments.
Alternatively, it is also possible that the labeling of the poly-
merase subunits with fluorescent proteins partially preclude
the interaction of these subunits with host nuclear import fac-
tors, thus impairing the potential coimport of PB2 and PB1 in
the nucleus. Our FCCS measurements showed that only a
minor portion of PB1 (28% and 10% in the cytoplasm and the
nucleus, respectively) appeared within a complex with PB2,
confirming the relatively weak interaction between PB1 and
PB2 already reported (9). It is thus likely that the strong in-
terface that can be formed between the N-terminal extremity
of PB2 and the C-terminal extremity of PB1 (45) is present
only in the fully assembled trimeric polymerase.
It was previously observed that coexpressed PA and PB2
improved nuclear accumulation of PB1 (12). However, only
the combined effect of coexpressed PB2 and PA was tested and
no comparison between the localization of PB1 in the presence
of PA only and in the presence of both PA and PB2 was
possible. In contrast, we compared triple coexpression (PB1,
PB2, and PA) with all combinations of double coexpression
and found that nuclear accumulation of coexpressed PB1 and
PA is significantly increased in the presence of PB2. Further-
more, we could show for the first time that not only the pres-
ence of PB2 but also its ability to be efficiently imported into
the nucleus is important, because the NLS-impaired mutant
PB2-QNQ did not increase the nuclear accumulation of PB1
and PA, although PB2-QNQ retained the ability to form a
trimeric complex. One possible explanation for the effect of
PB2 on the localization of PB1 and PA is the formation of the
heterotrimeric polymerase in the cytoplasm and its subsequent
nuclear import driven by the NLS of PB2. Although in the
FCCS experiments we did not detect any trimeric complexes in
the cytoplasm, we cannot exclude the possibility that cytoplas-
mic heterotrimers are very efficiently imported into the nucleus
after assembly and therefore their steady-state concentration
in the cytoplasm is kept low. However, nuclear import of fully
assembled stable trimers as the main route of transport for
influenza virus polymerase appears unlikely, because RanBP5
(8) and Hsp90 (30) efficiently compete with trimer formation
and because of the selective effect of Hsp90 inhibition on
nuclear import of PB1/PA but not PB2 (3). Furthermore, al-
though a minor fraction of PB1/PB2 dimers forms in the cyto-
plasm when these subunits are expressed alone, this did not
lead to increased nuclear accumulation of PB1, suggesting that
PB1 is not “carried” into the nucleus by the PB2 NLS. There-
fore, we favor a model where the PB1/PA dimer and PB2 have
independent transport routes into the nucleus (Fig. 6). Conse-
quently, formation of the trimeric polymerase would normally
occur in the nucleus and retain the subunits in the nucleus,
leading to an efficient nuclear accumulation of PB1 and PA.
The necessity to form heterotrimers in the nucleus to retain
PB1 and PA may contribute to the poor replication activity of
various “reassortant” polymerases composed of functionally
“normal” subunits originating from different strains that are
not optimally compatible in assembly (26).
Importin ?-mediated nuclear import of PB2 may prevent
aberrant formation of the trimeric polymerase in the cyto-
plasm. Competition between host factor binding and associa-
tion of polymerase subunits has been previously reported. For
instance, PB2 binds PB1/PA after the nuclear import factor
RanBP5 dissociates from PB1/PA dimer (9), and Hsp90 was
found bound to the PB1/PB2 dimer and PB2 alone, but this
binding was strongly reduced in the presence of PA, allowing
the formation of the PB2/PB1/PA heterotrimer (30). Here, we
show that mutating the PB2 NLS, which mediates binding to
importin alpha (46), leads to the aberrant presence of the
trimeric polymerase in the cytoplasm. This observation can be
interpreted in two different ways. (i) The binding of the wild-
type PB2 to importin alpha in the cytoplasm and its subsequent
rapid nuclear import could be a mechanism to prevent the
formation of the trimer in the wrong compartment. In the
nucleus, PB2 dissociation from the import factors by RanGTP
would then enable the formation of the correctly localized,
functional heterotrimer. For the PB2-QNQ mutant, its com-
promised interaction with the importin alpha in the cytoplasm
would induce the aberrant formation of the trimeric polymer-
ase in this compartment. (ii) Alternatively, the complex PB2-
QNQ/PA/PB1 could still form specifically in the nucleus, sim-
ilarly to the wild-type trimer, but then would leak or be
transported back into the cytoplasm. The efficient nuclear ac-
cumulation of PB2 would thus be required for the correct
localization of the trimeric polymerase inside the nucleus. To-
gether, these results emphasize the importance of the C-ter-
minal NLS of PB2, mutation of which impairs the nuclear
accumulation of all polymerase subunits. However, it should be
noted that the severe reduction of viral RNA replication for
the PB2-QNQ mutant may not be solely explained by impaired
nuclear accumulation, because restoration of nuclear localiza-
tion of PB2-QNQ by an alternative nuclear import mechanism
did not restore the replication efficiency (37).
The trimeric influenza virus polymerase forms higher-order
oligomers in the nucleus. Our photon-counting histogram ex-
periments showed that a fraction of the trimeric polymerase
complexes interact with each other in the nucleus to form
oligomers. Thus, we provide direct in vivo confirmation for the
homo-oligomerization of influenza virus polymerase, which
was recently suggested on the basis of in vitro studies of TAP-
purified polymerase subunits (20). It has been proposed that
the interactions between the polymerase present in the ribo-
nucleoprotein particles and the incoming polymerase molecule
may facilitate binding of the latter to the specific site at the 5?
terminus of the nascent viral RNA, thus promoting replication
and transcription of the viral RNA (21).
We also observed that the polymerase subunits, whether or
not they are in a complex with their counterparts, display
dramatically reduced mobility compared to that expected for
freely diffusing molecules of comparable size. This slow mo-
bility cannot be explained by the formation of higher-order
homo-oligomers, since single subunits and the PB1/PA dimer
only show a very limited tendency for homo-oligomerization.
Furthermore, the ?35% increase in molecular brightness ob-
served by PCH for the PA-mEGFP/PB1-mCFP/mCherry-PB2
complex in the nucleus compared to PA-mEGFP expressed
alone suggests that only a minor fraction of the polymerases
are forming higher-order oligomers. In contrast, all the poly-
1262 HUET ET AL.J. VIROL.
merases should be engaged in homo-oligomers of more than
100 polymerases to account for the very slow diffusion of the
PB2/PB1/PA complex. These observations imply that the poly-
merase subunits or combinations of them always exist within
large, slowly diffusing complexes with host factors. It is obvi-
ously important to identify these host factors or structures, as
they probably play an important role in the transport, assem-
bly, and function of the viral polymerase. Candidates are trans-
port factors such as HSP90 (30), importin ? (13), and RanBP5
(8), as well as RNA polymerase II (11, 36), the polyadenylation
machinery (24), and chromatin (14).
Conclusions. Taken together, our direct in vivo evidence
allows us to define a working model for influenza virus poly-
merase import and assembly. Our data show that the model
originally suggested by Deng and coauthors based on indirect
evidence (9) indeed applies to living cells. The PB1/PA het-
erodimer is imported into the nucleus by RanBP5, a member
of importin ? family (8). In the nucleus, PB1/PA associates
with PB2, which is imported independently by importin ? (13,
46) to form the fully assembled trimeric polymerase. We dem-
onstrate that NLS-mediated PB2 import is required to accu-
mulate PB1 and PA in the nucleus and prevent aberrant pres-
ence of the trimeric polymerase in the cytoplasm. These
insights illustrate the power of FCCS to monitor the interac-
tions of viral polymerase subunits in vivo. This method prom-
ises to be invaluable to study polymerase function and inter-
actions with host factors in the future, as well as to determine
the molecular consequences of host range mutations of influ-
We are grateful to S. Terjung, M. Wachsmuth, and C. Maeder for
technical assistance with the microscope used for FCCS experiments
and to Leica Microsystems for continuous support of the Advanced
Light Microscopy Facility at the European Molecular Biology Labo-
ratory (EMBL), Heidelberg. We also thank J. Ortin and N. Naffakh for
useful discussions and technical help.
S.H. and S.A. were supported by fellowships from the European
Molecular Biology Organization and from the EMBL Interdisciplinary
Postdocs Programme (EIPOD), respectively. L.F. was funded by the
ANR FLU INTERPOL contract (ANR-06-MIME-014-02).
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