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Annexin A6-Balanced Late Endosomal Cholesterol Controls Influenza
A Replication and Propagation
Agnes Musiol,
a
Sandra Gran,
a
Christina Ehrhardt,
b
Stephan Ludwig,
b
Thomas Grewal,
c
Volker Gerke,
a
Ursula Rescher
a
Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation and Interdisciplinary Clinical Research Centre, University of Münster, Münster, Germany
a
;
Institute of Molecular Virology, Centre for Molecular Biology of Inflammation, University of Münster, Münster, Germany
b
; Faculty of Pharmacy A15, University of Sydney,
Sydney, NSW, Australia
c
ABSTRACT Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope
lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection
cycle. We show that shifting of cellular cholesterol pools via the Ca
2ⴙ
-regulated membrane-binding protein annexin A6 (AnxA6)
affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and in-
crease late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6
ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus
propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replen-
ishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1).
Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV
replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to
cellular cholesterol homeostasis.
IMPORTANCE Influenza A virus (IAV) is a major public health concern, and yet, major host-pathogen interactions regulating IAV
replication still remain poorly understood. It is known that host cell cholesterol is a critical factor in the influenza virus life cycle.
The viral envelope is derived from the host cell membrane during the process of budding and, hence, equips the virus with a spe-
cial lipid-protein mixture which is high in cholesterol. However, the influence of host cell cholesterol homeostasis on IAV infec-
tion is largely unknown. We show that IAV infection success critically depends on host cell cholesterol distribution. Cholesterol
sequestration in the endosomal compartment impairs progeny titer and infectivity and is associated with reduced cholesterol
content in the viral envelope.
Received 2 August 2013 Accepted 8 October 2013 Published 5 November 2013
Citation Musiol A, Gran S, Ehrhardt C, Ludwig S, Grewal T, Gerke V, Rescher U. 2013. Annexin A6-balanced late endosomal cholesterol controls influenza A replication and
propagation. mBio 4(6):e00608-13. doi:10.1128/mBio.00608-13.
Invited Editor Stephan Pleschka, Justus-Liebig-University Giessen Editor Vincent Racaniello, Columbia University College of Physicians & Surgeons
Copyright © 2013 Musiol et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported
license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to Ursula Rescher, rescher@uni-muenster.de.
Influenza A virus (IAV) remains a major public health concern,
not only by causing thousands of deaths because of annual epi-
demics and rare but often severe pandemics but also by leading to
enormous economic loss every year (1). As agents directed against
viral components select for resistant mutants, new antiviral ther-
apeutic approaches might target the interaction of virus with host
cell components. Despite the enormous progress in influenza-
related research in the last decade, major host-pathogen interac-
tions regulating IAV replication and propagation still remain
poorly understood. The virus is characterized by a segmented,
single-stranded RNA genome with negative orientation, and its
genome encodes up to 12 viral structural and nonstructural pro-
teins (2). The virus genome is enclosed by an envelope that is
derived from the host cell membrane during the process of bud-
ding and, hence, equips the virus with a special lipid-protein mix-
ture. As this process is heavily dependent on the presence of spe-
cialized and cholesterol-enriched lipid microdomains, or so-
called “rafts,” this leads to a high level of cholesterol, a major
component of those raft domains, in the virus envelope (3–7).
Host cell cholesterol is a critical factor in IAV replication and
propagation. It is known that viral assembly and budding, as well
as infectivity, are strongly dependent on cellular cholesterol levels,
indicating the great importance of this host factor for virus infec-
tion (7–11). However, the molecular mechanisms of these regula-
tory interactions are largely unknown. This is partly due to the
limited knowledge about intracellular cholesterol transport be-
tween distinct membrane compartments in the host cell that reg-
ulates cholesterol homeostasis, as well as cholesterol-sensitive
protein trafficking (12).
Recently, annexin A6 (AnxA6) has emerged as an important
player in the maintenance of cellular cholesterol homeostasis (13–
16). Annexin A6 is a member of the annexin protein family of
structurally highly conserved, Ca
2⫹
-regulated membrane-
binding proteins that have been linked to the regulation of mem-
brane recognition and trafficking (17–20). All annexins share a
common structure composed of two domains: a conserved core
that is responsible for Ca
2⫹
and phospholipid binding and an
N-terminal tail that is unique for each annexin. Due to their role in
RESEARCH ARTICLE
November/December 2013 Volume 4 Issue 6 e00608-13 ®mbio.asm.org 1
membrane dynamics, annexins have already been shown to be
involved in the life cycles of several pathogens, including diverse
viruses. Regarding infections with IAV, proteomic analysis of in-
fluenza virions revealed the incorporation of annexins A1, A2, A4,
A5, and A11 into IAV particles (21). For AnxA2, it was even re-
ported that the protein has a supportive role for IAV replication
(22, 23). Recently, AnxA6 was proposed to be negatively involved
in IAV replication (24). Here, we elucidate the molecular mecha-
nism through which annexin A6 exerts a strong antiviral effect.
We show that AnxA6 affects the infectivity of progeny virus par-
ticles through shifting intracellular cholesterol pools. This effect
was independent of the plasma membrane-associated pool of
AnxA6 and could be reversed either through exogenous replen-
ishment of host cell cholesterol or by overexpression of the late
endosomal cholesterol transporter NPC1. These studies support a
role for AnxA6 in IAV replication and propagation and indicate
that cellular cholesterol homeostasis is critically linked to the in-
fectivity of the virus.
RESULTS
Annexin A6 negatively modulates influenza virus replication.
To examine the function of AnxA6 in IAV replication, we em-
ployed the human epithelial carcinoma cell line A431 (hereinafter
called A431wt, for A431 wild type), which naturally lacks endog-
enous AnxA6, and the A431-A6 cell line, which has stable expres-
sion of AnxA6 (25, 26). Western blot analysis of cell lysates con-
firmed that the expression of AnxA6 was only detectable in
A431-A6 cells (Fig. 1A, bottom). A431wt and A431-A6 cells were
infected with the avian IAV isolate A/FPV/Bratislava/79 (H7N7;
FPV) at a multiplicity of infection (MOI) of 0.01, and progeny
virus titers were monitored over a time period of 48 h (Fig. 1A).
The infectious titers of viruses produced by these cells and released
into the cell culture supernatants were measured by a standard
plaque assay technique. Both cell lines were permissive for IAV
replication; however, in A431-A6 cells, the virus titers were im-
paired at every time point analyzed. This correlated with a reduced
expression of virion-associated matrix protein 1 (M1), as assessed
by Western blotting (Fig. 1A, bottom).
To confirm this result in a cell model relevant for infections of
the upper respiratory tract and to exclude an aberrant phenotype
as a consequence of clonal selection during A431-A6 cell line gen-
eration, we repeated this experiment using human A549 lung ep-
ithelial cells transiently transfected with green fluorescent protein
(GFP)-tagged AnxA6 (A6-GFP) or GFP alone as a control. GFP
and A6-GFP expression were verified by fluorescence microscopy
(data not shown) and Western blotting (see Fig. S1 in the supple-
mental material). At 24 h after transfection, cells were infected as
described above. Again, virus titers were significantly reduced in
AnxA6-overexpressing cells (Fig. 1B). Furthermore, impaired vi-
ral progeny titers again correlated with reduced viral M1 protein
expression. This observation strengthened the finding that ele-
vated AnxA6 expression negatively influences IAV replication.
To further verify an involvement of AnxA6 in IAV replication,
we performed small interfering RNA (siRNA)-mediated knock-
down of AnxA6 in A549 cells using a pool of AnxA6-specific
siRNA duplexes and nontargeting siRNA as a control. At 48 h after
transfection, cells were infected with FPV at an MOI of 0.1, and
virus replication was allowed to proceed for 24 h. Efficient and
reproducible AnxA6 knockdown was confirmed by Western blot
analysis (see Fig. S1 in the supplemental material). In line with a
role of AnxA6 as a negative regulator, downregulation of AnxA6
resulted in significantly enhanced progeny virus titers in cell cul-
ture supernatants compared to the titers in control siRNA-treated
cells (Fig. 1B, top right). This correlated with higher M1 virus
protein expression.
Additional experiments revealed that AnxA6 also inhibited the
replication of other influenza strains, such as the H1N1 IAV strain
A/Puerto Rico/8/34 (PR8) and a mouse-adapted strain (H1N1v;
HH/04-3rd) of the 2009 pandemic swine-origin influenza A virus
FIG 1 AnxA6 negatively modulates influenza A virus replication. A431 wild-type (wt) and A431 cells stably overexpressing AnxA6 (A6) (A), as well as A549 cells
transiently overexpressing GFP or AnxA6-GFP (B, left), were infected with the avian IAV isolate A/FPV/Bratislava/79 (H7N7; FPV) at an MOI of 0.01. At the
indicated time points postinfection (p.i.), progeny virus titers were determined by standard plaque assay. IAV M1 and AnxA6 protein levels in cell lysates were
determined by Western blotting. Equal protein loading was verified by using
␣
-tubulin. (B, right) A549 cells transfected with AnxA6 siRNA (si_A6) or
nontargeting control siRNA (si_ct) were infected with FPV for 24 h at an MOI of 0.1, and viral progeny were determined by standard plaque assay. Levels ofM1
and AnxA6 proteins were monitored by Western blotting, and blots were probed for STAT3 to verify equal protein loading. Mean values ⫾SEM of at least three
independent experiments were calculated and assessed for statistically significant differences using a two-tailed ttest. *, Pⱕ0.05; **, Pⱕ0.01; ***, Pⱕ0.001.
Musiol et al.
2®mbio.asm.org November/December 2013 Volume 4 Issue 6 e00608-13
subtype H1N1 variant (H1N1v) strain A/Hamburg/04/2009 (see
Fig. S2 in the supplemental material).
Annexin A6 overexpression inhibits IAV particle production
and decreases virus infectivity. So far, our results indicated an
impact of cellular AnxA6 levels on IAV infection. To determine
whether the decrease in progeny virus titers induced by AnxA6
overexpression was due to reduced infectivity or a decreased
amount of infectious virus particles, we first performed hemag-
glutination (HA) assays to determine the levels of IAV particles
present in the sample. For this purpose, we infected A431wt and
A431-A6 cells with FPV at an MOI of 0.1, allowed the infection to
proceed for 24 h, and used the resulting supernatants for the HA
assay. As shown by the results in Fig. 2A, supernatants from in-
fected A431wt cells (FPV_wt) caused hemagglutination of red
blood cells up to the 1:32 dilution, whereas viral particles pro-
duced by A431-A6 cells (FPV_A6) needed the 1:16 dilution.
Therefore, the amount of viral particles produced by A431-A6
cells was decreased by approximately 50% compared to the
amount produced by controls. This strongly indicated that high
AnxA6 levels inhibit the production of viral particles per se. How-
ever, an additional effect on infectivity could not be excluded, as
the decrease in infectious titers in AnxA6-overexpressing cells ap-
peared to be greater than the 50% decrease in the total production
of IAV particles. To address the infectivity of IAV particles, we
therefore reinfected A549 cells with virus obtained from infected
A431wt (FPV_wt) or A431-A6 (FPV_A6) cells. To obtain one cy-
cle of IAV replication, we allowed the infection to proceed for 8 h.
Although A549 cells were infected with the same MOI of the re-
spective virus (MOI of 0.1), the virus titers were still decreased in
cells infected with virus that originated from A431-A6 cells
(Fig. 2B, left). Next, we investigated the entry process of the re-
spective virus into the host cell. Therefore, virion-associated ma-
trix protein was detected by Western blot analysis, as described
previously (27). We infected A549 cells with FPV_wt or FPV_A6
at an MOI of 5 and followed IAV internalization by detection of
viral M1 protein (Fig. 2B, right). A549 cells infected with FPV_A6
showed a strong decrease in the amount of M1 protein compared
to the amount in cells infected with FPV_wt, indicating that the
virus produced in A431-A6 cells displayed reduced capability in
the entry process and, hence, exhibited decreased infectivity of
viral particles.
In conclusion, high levels of AnxA6 not only decreased the
amount of viral particles produced by the host cell but, in addi-
tion, decreased the infectivity of those particles.
Plasma membrane-associated functions of AnxA6 are not re-
sponsible for its antiviral activity. Next, we aimed to investigate
FIG 2 AnxA6 upregulation decreases the amount and infectivity of viral particles. (A) A431wt and A431-A6 cells were infected with FPV (MOI of 0.1 for 24 h),
and supernatants obtained from either A431wt (FPV_wt) or A431-A6 (FPV_A6) cells were analyzed by hemagglutination assay. ⫹control, positive control; ⫺
control, negative control. Arrows indicate the greatest serum dilution giving a visible agglutination. (B) A431wt and A431-A6 cells were infected with FPV (MOI
of 0.1 for 48 h), and progeny virus titers were determined by standard plaque assay. A549 cells were then infected for 8 h with the virus replicated in A431wt
(FPV_wt) or A431-A6 cells (FPV_A6). For determination of infectious viral titers, an MOI of 0.1 was used. To monitor virus entry, A549 cells were infected with
an MOI of 5 for 15 to 360 min, as indicated. IAV M1 protein levels in cell lysates were monitored by Western blotting. Equal protein loading was verified using
␣
-tubulin. Mean values ⫾SEM of at least three independent experiments were calculated and assessed for statistically significant differences using a two-tailed
ttest. *, Pⱕ0.05.
Late Endosomal Cholesterol in IAV Infection
November/December 2013 Volume 4 Issue 6 e00608-13 ®mbio.asm.org 3
the underlying mechanism by which high AnxA6 levels decreased
the infectivity of IAV particles. Upon cell activation, AnxA6 binds
to negatively charged phospholipids that are found predomi-
nantly in the plasma membrane but also in endosomal mem-
branes. As AnxA6 is mainly localized at the plasma membrane, we
first addressed a role for plasma membrane-associated AnxA6 for
antiviral activity.
Recently, AnxA6 was found to target p120GAP and protein
kinase C
␣
(PKC
␣
) to the plasma membrane, thereby inactivating
Ras and epidermal growth factor receptor (EGFR), respectively,
and to downregulate the Raf/MEK/ERK (extracellular signal-
regulated kinase) signaling cascade (28–31). As this signaling
pathway is activated in a biphasic manner during IAV infections
and is essential for virus production (32, 33), high levels of AnxA6
might lead to an inhibition of IAV replication by disturbing Raf/
MEK/ERK signaling. To examine this possibility, we infected
A431wt and A431-A6 cells (FPV at an MOI of 5) and monitored
ERK1/2 activation by Western blotting. As shown by the results in
Fig. 3A, the kinetics of ERK1/2 phosphorylation during infection
proceeded in a similar manner in both cell lines.
Besides being a scaffold for EGFR and Ras signaling, AnxA6
exhibits further functions at the plasma membrane, as it associates
with cholesterol-rich membrane microdomains termed lipid rafts
and may function as an organizer of those domains to regulate
transient membrane-actin interactions during endocytosis. Fur-
thermore, AnxA6 has been shown to be involved in clathrin-
mediated endocytosis, which is exploited by IAV to enter host cells
(26, 34–37). To address whether AnxA6 might be involved in the
entry process of IAV into the host cell, we monitored the kinetics
of the appearance of the virion-associated matrix protein 1 (M1)
in A549 cells expressing GFP or A6-GFP following infection (FPV
at an MOI of 10). The M1 levels were comparable in AnxA6-
overexpressing cells and controls, indicating that high levels of
AnxA6 had no inhibitory effect on early stages of the viral life cycle
(from entry to escape from late endosomes) (Fig. 3B).
The results described above suggested that plasma membrane-
associated AnxA6 was not responsible for the antiviral activity. To
confirm this, A549 cells were transfected with A6-GFP and
membrane-anchored AnxA6-GFP (AnxA6-GFP-th, generated by
the addition of the complete H-Ras membrane targeting signal)
(16, 28, 38). GFP and plasma membrane-anchored GFP (GFP-th)
served as controls. At 24 h after transfection, the cells were infected
with FPV (MOI of 0.01 for 24 h). A6-GFP and A6-GFP-th expres-
sion levels were verified by fluorescence microscopy (data not
shown) and Western blotting (see Fig. S1B in the supplemental
material). As described above, the virus titers were decreased in
cells overexpressing AnxA6 but not in cells expressing AnxA6-th
compared to the titers in the respective control (Fig. 3C). Taken
together, AnxA6 is not likely to engage in antiviral activity by
interfering with virus entry and virus-induced mitogen-activated
protein kinase (MAPK) signaling at the plasma membrane.
High levels of AnxA6 lead to cholesterol sequestration in
A549 cells. Host cell cholesterol is a critical factor in IAV replica-
tion. Viral assembly and budding, as well as infectivity, are
strongly dependent on cellular cholesterol distribution, indicating
the great importance of this host factor for virus infection (3–5, 7).
However, the molecular mechanisms underlying the link between
cellular cholesterol and virus replication are largely unknown. Re-
cently, AnxA6 was proposed to be involved in the regulation of
cholesterol homeostasis: high levels of AnxA6 were shown to in-
duce an NPC1-like phenotype, as characterized by an accumula-
tion of cholesterol in late endosomes. Inhibition of cholesterol
export from the late endocytic compartment in AnxA6-expressing
cells was associated with reduced cholesterol levels in the Golgi
apparatus and the plasma membrane (15, 16). These findings
prompted us to investigate whether modulation of cholesterol ho-
meostasis by AnxA6 could be responsible for the AnxA6-mediated
inhibition of IAV replication.
To address this, we first compared the cholesterol distribution
in AnxA6-overexpressing A549 cells and controls, as described
previously (15, 16). A549 cells were transfected with A6-GFP or
GFP, fixed, stained with filipin, and analyzed for their cellular
cholesterol distribution by confocal microscopy. Treatment with
U18666A, a hydrophobic polyamine known to promote the accu-
mulation of cholesterol in late endosomes, served as a positive
control. In control cells, cholesterol was detectable at the plasma
membrane and in punctate structures throughout the cytoplasm.
In contrast, the majority of A6-GFP-overexpressing cells showed a
very different staining pattern. In particular, a much stronger ac-
cumulation of cholesterol in mostly perinuclear vesicles was ob-
served (Fig. 4A). This accumulation resembled the scenario in
U18666A-treated cells, indicating that the cholesterol accumula-
tion in late endosomes observed previously in several AnxA6-
overexpressing cell lines (15, 16) also holds true for AnxA6 over-
expression in A549 cells.
Cholesterol accumulation in late endosomes is responsible
for the inhibition of virus replication by AnxA6. We next inves-
tigated whether U18666A-induced late endosomal cholesterol ac-
cumulation could interfere with IAV propagation. Therefore,
A549 cells were treated with and without U18666A overnight and
infected with FPV at an MOI of 0.1, and virus replication was
allowed to proceed for 24 h. The infectious titers of viruses pro-
duced in cell culture supernatants were measured by a standard
plaque assay technique. Consistent with a model of inhibition of
cholesterol egress from late endosomes blocking virus propaga-
tion, U18666A treatment and AnxA6 overexpression had strik-
ingly similar inhibitory effects on infectious progeny virus titers
(ca. 50%) (compare Fig. 4B and 2A). This correlated with the
reduced expression of viral M1 protein in U18666A-treated A549
cells (Fig. 4B, bottom).
To further substantiate these findings, the progeny virus titers
of A549 cells preincubated with and without U18666A overnight,
infected with IAV at an MOI of 0.1 for 24 h, and ectopically ex-
pressing A6-GFP or GFP were compared. Consistent with the re-
sults described above, U18666A treatment significantly impaired
the progeny virus titers of GFP-expressing A549 cells, and the M1
protein levels were downregulated in these cells (Fig. 4B). In
AnxA6-GFP-overexpressing A549 cells, however, the addition of
U18666A had only a minor inhibitory effect on infectious virus
particles and M1 protein expression (Fig. 4C). Taken together, the
inhibition of cholesterol export from late endosomes by U18666A
treatment leads to significantly reduced progeny virus titers,
strongly suggesting that the inhibitory effect of AnxA6 overex-
pression on IAV replication is due to an inhibition of cholesterol
egress from late endosomes.
Restoration of cellular cholesterol balance in AnxA6-
overexpressing cells restores influenza virus replication. AnxA6
is recruited to late endosomes in a cholesterol-dependent manner
(13) and, possibly, through physical interaction with NPC1,
which could block NPC1-dependent cholesterol export from the
Musiol et al.
4®mbio.asm.org November/December 2013 Volume 4 Issue 6 e00608-13
FIG 3 IAV entry and MAPK signaling are not altered in AnxA6-overexpressing cells. (A) A431wt and A431-A6 cells were starved in 1% FBS overnight and
infected with FPV at an MOI of 5. Cells were lysed after 15 to 360 min, as indicated, and analyzed for total (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2)by
Western blotting. (B) A549 cells transiently overexpressing GFP or AnxA6-GFP were infected with FPV at an MOI of 10 for 0 to 240 min, as indicated. After cell
lysis, expression of IAV M1 protein was monitored by Western blotting and quantified. Mean values ⫾SEM were calculated from three independent experi-
ments.

-Actin served as a loading control. (C) A549 cells transiently overexpressing GFP, AnxA6-GFP, or plasma membrane-anchored GFP (GFP-th) or
AnxA6-GFP (AnxA6-GFP-th) were infected with FPV (MOI of 0.01 for 24 h), and progeny virus titers were determined by standard plaque assay. Mean values
⫾SEM of at least three independent experiments were calculated and assessed for statistically significant differences using one-way ANOVA followed by
Dunnett’s multiple comparison test. *, Pⱕ0.05; n.s., not significant.
Late Endosomal Cholesterol in IAV Infection
November/December 2013 Volume 4 Issue 6 e00608-13 ®mbio.asm.org 5
late endosomal/lysosomal compartment (15). Moreover, NPC1
overexpression restored the cellular cholesterol balance in AnxA6-
overexpressing cells (15, 16). Given the findings described above,
we reasoned that NPC1 overexpression could overcome the inhib-
itory effect of AnxA6 on viral replication. Therefore, A431-A6 cells
were transiently transfected with an expression vector encoding
yellow fluorescent protein (YFP)-tagged wild-type NPC1 (NPC1-
YFP). NPC1-YFP expression was verified in duplicate samples by
fluorescence microscopy (data not shown). At 24 h after transfec-
tion, cells were infected with FPV at an MOI of 0.1, and the infec-
tious titers were measured with a standard plaque assay technique
24 h postinfection (p.i.). In support of our hypothesis, the over-
expression of wild-type NPC1 partially restored progeny virus ti-
ters in A431-A6 cells (Fig. 5A). To further underscore this finding,
we analyzed viral replication upon overexpression of the loss-of-
function NPC1 P692S mutant (having a change of proline to ser-
ine at position 692), which cannot bind cholesterol and inhibits
cholesterol export from late endosomes (39–41). Indeed, while
IAV replication can be rescued by overexpression of wild-type
NPC1, the P692S mutant was not able to reestablish viral titers in
A431-A6 cells (Fig. 5B). P962S also significantly impaired IAV
replication in A431wt cells (not shown), further suggesting that
cholesterol pools from late endosomes are required for efficient
virus replication and propagation.
Inhibition of late endosomal cholesterol export reduces cho-
lesterol levels in other cellular compartments, such as the plasma
membrane. We speculated that the reduction of cholesterol levels
at the plasma membrane triggered by AnxA6, U18666A, or the
NPC1 mutant could be responsible for reducing viral propaga-
tion. To address this possibility, A549 cells were transiently trans-
FIG 4 Pharmacological inhibition of late endosomal cholesterol egress inhibits influenza virus replication in A549 cells. (A) A549 cells transiently overexpress-
ing GFP (green, left) or AnxA6-GFP (green, right) were treated with U18666A overnight. Cellular cholesterol was stained using filipin (blue). Bar, 20
m. (B and
C) A549 wild-type cells (B) or A549 cells transiently overexpressing GFP or AnxA6-GFP (C) were treated with U18666A overnight and then infected with FPV
(MOI of 0.1 for 24 h). Progeny virus titers were determined by standard plaque assay. IAV M1 protein and AnxA6 expression levels were monitored by Western
blotting. Equal protein loading was verified using STAT3 or
␣
-tubulin, as indicated. Mean values ⫾SEM of at least three independent experiments were
calculated and assessed for statistically significant differences using a two-tailed ttest. *, Pⱕ0.05; **, Pⱕ0.01.
Musiol et al.
6®mbio.asm.org November/December 2013 Volume 4 Issue 6 e00608-13
fected with A6-GFP or GFP, followed by the addition of exoge-
nous cholesterol to replenish plasma membrane cholesterol. Next,
cholesterol-treated and nontreated A549 cells were infected with
FPV at an MOI of 0.1, and the infectious titers were measured with
the standard plaque assay technique. Indeed, cholesterol replen-
ishment completely restored the progeny virus titers in A6-GFP-
overexpressing A549 cells (Fig. 5C), further supporting a role
for AnxA6 to modulate cholesterol-dependent steps during viral
replication. In conclusion, restoration of the cellular cholesterol
balance via cholesterol replenishment using exogenous choles-
terol or the ectopic expression of wild-type NPC1 in AnxA6-
overexpressing cells improves the ability of IAV to replicate and
propagate.
IAV cholesterol content is decreased in viral progeny re-
leased from cholesterol-imbalanced host cells. The data de-
scribed above strongly indicated a role for cholesterol in the anti-
viral effect of elevated AnxA6 contents. Budding from cholesterol-
rich sites at the plasma membrane provides the virus with a
cholesterol-rich envelope (6, 42). Hence, we assessed whether al-
tered cholesterol distribution in A431-A6 cells had an impact on
the cholesterol levels in the viral envelope. We therefore compared
the cholesterol contents of purified IAV released from A431 and
A431-A6 cells at 24 h p.i. The purity of IAV preparations was
controlled by verifying the absence of the viral nonstructural pro-
tein NS1 in Western blots (Fig. 6A). As shown by the results in
Fig. 6B, comparable amounts of cholesterol were detected in the
lysates of both cell lines before and after infection. However, virus
particles released from A431-A6 cells displayed a 50% reduction
in cholesterol content, indicating that cholesterol sequestration in
late endosomes and the concomitant decrease in cholesterol at the
cell periphery leads to less cholesterol being available for viral
budding and envelope formation.
DISCUSSION
The dynamics of membrane events and signaling are intimately
linked to the interactions of proteins with lipids and lipid domains
(43). Thus, many pathogens, including viruses, employ host cell
lipid-enriched microdomains at different points of their infection
process to efficiently infect the target cell (44). Like other envel-
oped viruses, IAV depends on the host membrane and its dynam-
FIG 5 Restoration of late endosomal cholesterol export restores IAV titers. (A and B) A431-A6 cells transiently expressing NPC1-YFP (A) or NPC1 P692S-YFP
(B) were infected with FPV (MOI of 0.1 for 24 h). Progeny virus titers were determined by standard plaque assay. (C) A549 cells transiently overexpressing GFP
or AnxA6-GFP were infected with FPV (MOI of 0.1 for 24 h). Exogenous cholesterol was added to the medium at 2 h p.i. Progeny virus titers were determined
by standard plaque assay and expressed as percentages of virus titers released from GFP-expressing control cells. Statistically significant differences of the mean
values ⫾SEM calculated from at least three independent experiments were assessed by two-tailed ttest (A, B) or by one-way ANOVA followed by Tukey’s
multiple comparison test (C). *, Pⱕ0.05; **, Pⱕ0.01; ***, Pⱕ0.001; n.s., not significant.
FIG 6 High levels of AnxA6 significantly decrease the cholesterol content in the viral envelope. A431wt and A431-A6 cells were infected with FPV (MOI of 0.1)
or mock treated with infection medium. At 24 h p.i., supernatants were concentrated, purified, and assayed, together with the respective cell lysates, for their
cholesterol content. (A) Purity of IAV preparations was verified by the exclusion of the viral nonstructural protein NS1 from virus samples in Western blots. (B)
Cholesterol content in cell lysates and viral particles (IAV) released from A431wt (wt) and A431-A6 cells (A6) before and after (p.i.) infection. Statistically
significant differences of the mean values ⫾SEM calculated from at least three independent experiments were assessed by Student’s ttest. **, Pⱕ0.01; n.s., not
significant.
Late Endosomal Cholesterol in IAV Infection
November/December 2013 Volume 4 Issue 6 e00608-13 ®mbio.asm.org 7
ics at several stages of the viral life cycle. Annexins constitute a
family of Ca
2⫹
-dependent host cell membrane proteins that have
different lipid specificities and, thus, associate with different target
membranes in the cell (17–19, 45). Annexins have already been
shown to act in viral infections (46), and IAV carries several an-
nexins in its particle, most likely as a consequence of budding at
raftlike domains enriched with annexins (22, 23).
Recently, AnxA6 has been proposed to negatively regulate IAV
infection through interaction with the IAV matrix protein M2
(24). Here, we show that AnxA6 levels in the host cell negatively
correlate with IAV replication and reveal that aberrant cholesterol
accumulation in late endosomes, reminiscent of an NPC1
mutant-like phenotype, in AnxA6-expressing cells causes antiviral
activity. Consistently, when cells were treated with the hydropho-
bic polyamine U18666A, a drug commonly used to mimic the
abnormal accumulation of unesterified cholesterol seen in late
endosomes of NPC1 mutant cells (47), a strong reduction in virus
titers was observed. These findings suggest that AnxA6 interferes
with NPC1-dependent cholesterol trafficking. In accordance
with this, increased expression of wild-type NPC1, known to
correct the NPC1 mutant-like phenotype in AnxA6-expressing
cells (15, 16), significantly improved the virus titers in AnxA6-
overexpressing cells. Cellular cholesterol is synthesized de novo in
the endoplasmic reticulum (ER) and transported to the plasma
membrane independently of NPC1 (47). Subsequently, it reinter-
nalizes to the ER or other cellular compartments and/or recycles
back to the plasma membrane. Dysfunctional NPC1 disturbs the
intracellular distribution of cholesterol, leading to its accumula-
tion in late endosomes and a secondary reduction of cholesterol
levels in other cellular sites, such as the Golgi apparatus and the
plasma membrane. This is also observed in AnxA6-
overexpressing cells (15). The fact that AnxA6 coimmunoprecipi-
tates with NPC1 suggests that a direct interaction of these proteins
in the late endocytic compartment interferes with the ability of
NPC1 to bind and transfer cholesterol across the late endosomal/
lysosomal membrane. Our finding that exogenous cholesterol
partially reversed the inhibitory effect of AnxA6 on IAV propaga-
tion indicates that cellular cholesterol trafficking from the late
endosome to other sites, most likely the plasma membrane, is
required for efficient IAV replication. These results also argue
against a major impact of other lipids, such as sphingosine, that
have been found to accumulate abnormally together with choles-
terol in the endo-/lysosomes of NPC mutant cells (48–50).
Over the past decade, cholesterol has been shown to be a cru-
cial host factor for IAV. It is assumed that this essential host mem-
brane component plays a decisive role during virus replication
(10, 11, 42). Within the host cell membrane, cholesterol functions
in intracellular transport and cell signaling, acting through lipid
rafts. These cholesterol-enriched membrane microdomains have
been implicated in several steps of the viral life cycle, including the
assembly and budding of progeny virus particles at the plasma
membrane (42). However, experimental evidence for the impor-
tance of rafts in IAV replication is mainly derived from detergent
extraction experiments, which drastically change the distribution
and potential clustering of certain lipids and proteins. More-
conclusive evidence that plasma membrane rafts are involved in
HA clustering is drawn from recent improvements in fluorescence
microscopy techniques that allowed the analysis of protein-raft
association in a more-physiological cellular membrane environ-
ment (51).
In contrast to other enveloped viruses that depend on host cell
components to ensure budding and the release of viral progeny,
IAV uses the virus-encoded M2 protein to mediate scission of
buds (52). Although IAV is thought to bud from cholesterol-rich
membrane domains, M2 was shown to partition into rafts only
when clustered with HA (51). Physical association of AnxA6 with
M2 has been reported previously (24) and was proposed to impair
IAV replication in AnxA6-overexpressing cells. However, our data
strongly suggest that AnxA6-mediated changes in cholesterol ho-
meostasis also have to be considered, as the restoration of cellular
cholesterol distribution, through NPC1 overexpression or the ad-
dition of exogenous cholesterol, reversed impaired IAV replica-
tion in AnxA6-overexpressing cells. This may point at AnxA6 ex-
erting multiple functions in different cellular locations that either
directly (M2) or indirectly (cholesterol) inhibit viral replication
and propagation. It is tempting to speculate that the drop in virus
titers observed in the previous study (24) was also accompanied by
major alterations in cholesterol distribution caused by AnxA6
up-/downregulation, as described here. As AnxA6 displays en-
hanced binding to membranes with elevated cholesterol levels (13,
26, 53, 54), the interaction of AnxA6 with M2 could serve to facil-
itate M2 targeting to the IAV bud zone to ensure the assembly of
virus components.
Host membrane-derived IAV envelope is enriched in lipids
generally found in raft microdomains, including cholesterol. In
fact, 44% of the total virus lipid is cholesterol, which represents
approximately 12% of the total mass of the virion (6, 42). Budding
of virus particles through rafts equips the particle with an appro-
priate lipid mixture that protects particles from environmental
damage and, in the case of cholesterol, might promote membrane
fusion upon virus entry. Hence, virus treated with cholesterol-
depleting agents shows reduced infectivity (10). Our results now
demonstrate that the AnxA6-mediated endosomal cholesterol se-
questration that leads to reduced cholesterol contents in the
plasma membrane (15) is associated with strongly reduced IAV
cholesterol contents and impaired infectivity.
A precise understanding of the molecular mechanisms that
underlie virus-host cell interactions is a prerequisite for targeting
host cell components and could open up efficient therapeutic
strategies. Antiviral drugs could circumvent the time-consuming
vaccine development and the viral resistance due to rapid anti-
genic mutation. Host cell factor targeting has recently emerged as
a promising approach (reviewed in reference 55), and recent find-
ings strongly suggest that modulating the host immune response
reduces mortality rates (56, 57). Thus, combination therapy of
two or more antiviral drugs with different modes of action (i.e.,
targeting the virus and the host cell) to prevent and control acute
infection could become the therapeutic approach of choice not
only for IAV but also for other microbial infections. Statin treat-
ment of hospitalized influenza patients is associated with reduced
mortality (58), although it is difficult to distinguish between the
immunosuppressive and the cholesterol-lowering effects. Target-
ing host cell components could provide an elegant approach to
dissect and functionally address the influence of cellular choles-
terol levels on IAV infection. Collectively, our data provide evi-
dence that host cell factors, such as AnxA6, involved in maintain-
ing proper cholesterol homeostasis have a major impact on IAV
replication. We conclude that AnxA6 indirectly regulates IAV rep-
lication by reducing the availability of cholesterol at the plasma
membrane, thereby equipping the budding virus with an envelope
Musiol et al.
8®mbio.asm.org November/December 2013 Volume 4 Issue 6 e00608-13
that is strongly reduced in cholesterol. Thus, targeting the cellular
cholesterol balance might ameliorate IAV infection. It remains to
be determined whether additional defects in the delivery and/or
assembly of viral components and cell surface molecules engaged
in influenza release contribute to the reduced IAV replication.
MATERIALS AND METHODS
Cells, viruses, and infection conditions. The human alveolar epithelial
cell line A549, the human epithelial carcinoma cell line A431 (A431wt),
and A431-derived A431-A6 cells were cultivated in Dulbecco’s modified
Eagle’s medium (DMEM). The generation of stable AnxA6-expressing
A431 (A431-A6) cells has been described previously (31). Madin-Darby
canine kidney (MDCK) cells were cultured in minimal essential medium
(MEM). Cell culture media were supplemented with 10% heat-
inactivated fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml strep-
tomycin. All cell lines were cultured at 37°C in a humidified 5% CO
2
atmosphere.
The avian influenza virus A/FPV/Bratislava/79 (H7N7; FPV) and the
human prototype strain A/Puerto Rico/8/34 (H1N1) (PR8) were origi-
nally obtained from the virus strain collection of the Institute of Virology,
Giessen, Germany. The mouse-adapted S-OIV A/Hamburg/04/2009
(H1N1v) strain (H1N1v; HH/04-3rd), adapted to efficient propagation in
mice by sequential lung-to-lung passages, was generated in house (59). All
viruses were propagated in MDCKII cells. For infection, cells were washed
with phosphate-buffered saline (PBS) and incubated with the respective
virus at the indicated multiplicities of infection (MOI) diluted in PBS-BA
(PBS containing 0.2% bovine serum albumin [BSA; MP Biomedicals],
1 mM MgCl
2
, 0.9 mM CaCl
2
, 100 U/ml penicillin, and 0.1 mg/ml strep-
tomycin) at 37°C. After 30 min, the inoculum was aspirated, and cells were
washed with PBS and incubated with DMEM-BA (DMEM containing
0.2% BSA, 1 mM MgCl
2
, 0.9 mM CaCl
2
, 100 U/ml penicillin, and
0.1 mg/ml streptomycin) for the times indicated.
Plaque titration. To quantify virus production, supernatants of in-
fected cells were collected at the indicated times postinfection (p.i.) in
duplicate experiments to assess the number of infectious particles by a
standard plaque assay technique. For this purpose, MDCK cells grown to
a monolayer in six-well dishes were washed with PBS and infected with
serial dilutions of the respective supernatants in PBS-BA for 30 min at
37°C. The inoculum was replaced with 2 ml MEM-BA (MEM containing
0.2% BSA, 1 mM MgCl
2
, 0.9 mM CaCl
2
, 100 U/ml penicillin, and
0.1 mg/ml streptomycin) containing 0.6% agar (Oxoid, Hampshire,
United Kingdom), 0.3% DEAE-dextran (Amersham Pharmacia Biotech,
Freiburg, Germany), and 1.5% NaHCO
3
(Gibco Invitrogen, Karlsruhe,
Germany) and incubated at 37°C. After 2 days, virus plaques were visual-
ized by staining with neutral red. Virus titers were depicted as PFU/ml.
HA assay. Hemagglutination (HA) assays were performed in
V-bottomed microtiter plates. Briefly, serial 2-fold dilutions of virus su-
pernatants in PBS were prepared in microtiter plates in a volume of 50
l.
Additionally, PBS was used as a negative control and purified virus as a
positive control. Amounts of 50
l of chicken erythrocytes were added to
the wells and were analyzed following1hofincubation at 4°C. Hemag-
glutination was observed with the unaided eye and monitored by photog-
raphy.
Virus purification. For purification of IAV particles, harvested cell
culture supernatants were first clarified by centrifugation (10 min at 700
⫻g) and then concentrated by using Centricon plus 70 filter devices
(Millipore). For this purpose, the filter devices were coated with 1 mg/ml
BSA overnight prior to virus concentration according to the manufactur-
er’s instructions.
Cholesterol quantification. The cholesterol contents in cell lysates
and IAV preparations were measured by using the Amplex red cholesterol
assay kit (Invitrogen) according to the manufacturer’s protocol. The re-
sults were normalized to total cellular protein.
Transient transfections, plasmids, and siRNAs. A549 and A431 cell
lines were transfected with plasmid or siRNA using Lipofectamine 2000
(Invitrogen) according to the manufacturer’s protocol. Human AnxA6
was expressed from the plasmid pEGFP-N1 (38), and murine NPC1 was
expressed from the plasmid pEYFP-N2 (39). pEGFP-N3 served as a con-
trol. AnxA6 fused to the H-Ras membrane anchor was expressed from the
pC1-based GFP-th plasmid. The cloning is described in detail in reference
38. Transfected cells were incubated for 24 h before the start of experi-
ments. Transfection efficiency was controlled by fluorescence micros-
copy, as well as by detection of the respective proteins with Western blot-
ting. For knockdown of AnxA6 protein expression, siRNA against human
AnxA6 (siGENOME SMART pool, human ANXA6; Dharmacon) was
used. Nontargeting siRNA (ON-Target plus siControl; Dharmacon)
served as a negative control. Transfected cells were incubated for 48 h
before the start of experiments, and transfection efficiency was controlled
by using Western blots.
Cell lysis and Western blotting. After infection for the indicated
times, cells were washed and harvested in 1.5 ml PBS and subsequently
pelleted by centrifugation (15,000 ⫻gfor 1 min), resuspended in an
appropriate amount of 8 M urea, and sonified. The protein concentration
in the lysates was determined by the Bradford method. Cell lysates were
used for protein expression analysis by SDS-PAGE and Western blotting.
The primary antibodies used for detection of the respective proteins were
mouse anti-influenza M1 monoclonal antibody (MAb) (AbD Serotec),
mouse anti-annexin A6 MAb (BD Transduction Laboratories), mouse
anti-
␣
-tubulin MAb (Sigma-Aldrich), rabbit anti-GFP polyclonal anti-
body (PAb) (Invitrogen), rabbit anti-

-actin PAb (Sigma-Aldrich), rabbit
anti-STAT3 MAb (Cell Signaling), mouse anti-MAPK p44/42 MAb
(L34F12; Cell Signaling), and rabbit anti-phospho-MAPK p44/42 MAb
(Thr202/Tyr204, D13.14.4E; Cell Signaling). Rabbit anti-AnxA6 PAb was
prepared in our laboratory and has been described elsewhere (13, 26).
IRDye secondary antibodies (LI-COR) labeled with near infrared (NIR)
fluorescent dyes used for direct, nonenzymatic detection of primary anti-
bodies were as follows: IRDye 680CW donkey anti-mouse IgG (H⫹L),
IRDye 800CW donkey anti-mouse IgG (H⫹L), IRDye 680CW donkey
anti-rabbit IgG (H⫹L), and IRDye 800CW donkey anti-rabbit IgG
(H⫹L). The Odyssey infrared imaging system (LI-COR) was used for NIR
fluorescence detection.
Quantification of Western blots. Western blots were quantified using
the Odyssey infrared imaging system software version 3.0.25. The total
band densities were measured against the local background. M1 signal
intensities were normalized to

-actin. All data are expressed as the means
of three independent transfection and infection experiments.
Filipin staining and microscopy. A549 cells destined for fluorescence
microscopy were fixed with 4% paraformaldehyde (PFA)-PBS for 10 min
at room temperature. For visualization of free cholesterol, fixed cells were
blocked with 2% BSA for 30 min, incubated with filipin (filipin complex
from Streptomyces filipinensis, diluted 1:50 in heat-inactivated fetal calf
serum; Sigma-Aldrich), and washed with PBS. Confocal microscopy was
carried out using an LSM 710 META microscope (Carl Zeiss, Jena, Ger-
many) equipped with a Plan-Apochromat 63⫻/1.4 oil immersion objec-
tive.
Treatment with exogenous cholesterol or U18666A. For cholesterol
replenishment experiments, water-soluble cholesterol (45 mg cholesterol
complexed with 955 mg methyl-

-cyclodextrin [M

CD]; Sigma) was
used. In brief, cholesterol was premixed for 60 min in DMEM-BA (30
g/
ml) and added to cells to a final concentration of 5
g/ml 2 h after infec-
tion. For the accumulation of cholesterol in late endosomes, cells were
treated for 16 h with 2
g/ml U18666A (Biomol). If the cells were used for
virus experiments, the infection of cells was followed by renewed treat-
ment with 2
g/ml U18666A. Filipin staining was used to control the late
endosomal accumulation of free cholesterol upon U18666A treatment.
Statistical analysis. All experiments were performed at least three
times, and mean values ⫾standard errors of the means (SEM) were cal-
culated. Statistical significance was evaluated by two-tailed ttest or by
one-way analysis of variance (ANOVA) followed by either Tukey’s or
Late Endosomal Cholesterol in IAV Infection
November/December 2013 Volume 4 Issue 6 e00608-13 ®mbio.asm.org 9
Dunnett’s multiple comparison test. A Pvalue of ⬍0.05 indicated a sta-
tistically significant difference.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://mbio.asm.org
/lookup/suppl/doi:10.1128/mBio.00608-13/-/DCSupplemental.
Figure S1, TIF file, 0.2 MB.
Figure S2, TIF file, 0.3 MB.
ACKNOWLEDGMENTS
This work was supported by grants from the Interdisciplinary Clinical
Research Center (IZKF; grant RE2/017/10) and the German Research
Foundation (GRK 1409, RE2611/2-1, SFB 1009/A6, and SFB 629/A1) to
U.R. and V.G. T.G. acknowledges support from the National Health and
Medical Research Council of Australia (NHMRC; grant 510294) and the
University of Sydney (grant 2010-02681).
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