Intact sphingomyelin biosynthetic pathway is essential for
intracellular transport of influenza virus glycoproteins
The MIT Faculty has made this article openly available. Please share
how this access benefits you. Your story matters.
Citation
Tafesse, F. G., S. Sanyal, J. Ashour, C. P. Guimaraes, M.
Hermansson, P. Somerharju, and H. L. Ploegh. “Intact
sphingomyelin biosynthetic pathway is essential for intracellular
transport of influenza virus glycoproteins.” Proceedings of the
National Academy of Sciences 110, no. 16 (April 16, 2013):
6406-6411.
As Published
http://dx.doi.org/10.1073/pnas.1219909110
Publisher
National Academy of Sciences (U.S.)
Version
Final published version
Accessed
Thu May 26 22:53:36 EDT 2016
Citable Link
http://hdl.handle.net/1721.1/84947
Terms of Use
Article is made available in accordance with the publisher's policy
and may be subject to US copyright law. Please refer to the
publisher's site for terms of use.
Detailed Terms
Intact sphingomyelin biosynthetic pathway is essential
for intracellular transport of influenza virus
glycoproteins
Fikadu G. Tafessea, Sumana Sanyala, Joseph Ashoura, Carla P. Guimaraesa, Martin Hermanssonb, Pentti Somerharjub,
and Hidde L. Ploegha,1
a
Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, MA 02142; and bDepartment of Biochemistry
and Developmental Biology, Institute of Biomedicine, University of Helsinki, 00014, Helsinki, Finland
Edited by Kai Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, and approved March 4, 2013 (received for review
November 14, 2012)
Cells genetically deficient in sphingomyelin synthase-1 (SGMS1) or
blocked in their synthesis pharmacologically through exposure to
a serine palmitoyltransferase inhibitor (myriocin) show strongly
reduced surface display of influenza virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). The transport of HA to the
cell surface was assessed by accessibility of HA on intact cells to
exogenously added trypsin and to HA-specific antibodies. Rates of
de novo synthesis of viral proteins in wild-type and SGMS1deficient cells were equivalent, and HA negotiated the intracellular
trafficking pathway through the Golgi normally. We engineered
a strain of influenza virus to allow site-specific labeling of HA and
NA using sortase. Accessibility of both HA and NA to sortase was
blocked in SGMS1-deficient cells and in cells exposed to myriocin,
with a corresponding inhibition of the release of virus particles from
infected cells. Generation of influenza virus particles thus critically
relies on a functional sphingomyelin biosynthetic pathway, required to drive influenza viral glycoproteins into lipid domains of
a composition compatible with virus budding and release.
lipid raft
| flu assembly | sortagging | virus–host interaction
R
elease of newly assembled influenza virions requires not only
a proper mixture of the viral constituents themselves but
also relies on host factors such as endoplasmic reticulum (ER)
components, glycosyltransferases, and factors that determine intracellular vesicular transport. Some of these host factors might
serve as targets of new antiviral strategies (1). Given the tendency
of viruses to acquire resistance under selective pressure exerted
by the immune system or imposed by antiviral drugs (2–4), host
factors are more likely to escape the emergence of resistance.
Genome-wide RNAi screens designed to identify such host factors are predicated on this notion, as exemplified for HIV and
influenza virus in several recent reports (5–7). These types of
genetic approaches readily establish the involvement of particular
proteins or other template-encoded products in viral biogenesis.
In contrast, the role of complex carbohydrates and lipids is more
difficult to discern, precisely because these are not themselves
template-encoded, and rather are the end products of complex
biosynthetic pathways. Although it is straightforward to alter by
mutation a viral nucleic acid and the products it encodes, it
is more difficult to selectively modify the carbohydrate or lipid
composition of a virus without affecting many host functions in
the process.
Enveloped viruses incorporate host components into their
membranes for the construction of new infectious particles. The
lipid composition of newly formed virions resembles that of the
membrane compartments from which they bud. Vesicular stomatitis virus and influenza virus, which bud from the basolateral
and apical domains of polarized epithelial cells, respectively, report on the lipid composition of these membrane domains (8, 9).
The targeting signals carried by viral membrane glycoproteins,
together with the propensity of viral membrane proteins to
6406–6411 | PNAS | April 16, 2013 | vol. 110 | no. 16
interact preferentially with certain classes of (glyco)sphingolipids,
help determine the characteristics of the membrane domain(s)
from which new particles originate (8).
The influenza virus envelope contains two major membrane
glycoproteins, hemagglutinin (HA) and neuraminidase (NA),
along with the M2 protein (10, 11). Assembly of new virus particles
requires the coalescence of HA and NA in membrane domains of
distinct lipid composition, with M2 proposed to play a later role in
membrane scission (12) to achieve particle release. High local
concentrations of cholesterol, sphingomyelin (SM), and glycosphingolipids impart on these membrane microdomains—also
called “rafts”—unique biophysical properties, such as enhanced
resistance to extraction by nonionic detergents (13). The debate
about the physical size, lifetime, and dynamic behavior of these
lipid rafts is unlikely to subside anytime soon (14, 15), nor is it
clear whether these are the only membrane specializations controlled by lipid composition.
The role of lipid rafts in cellular processes is a matter of record
(16, 17), and perturbation of the homeostasis of its major component, cholesterol, affects membrane behavior (14). Physical
extraction of cholesterol with methyl-β-cyclodextrin and/or treatment of cells with drugs that inhibit cholesterol metabolism are
common strategies used to perturb cellular pools of cholesterol
(18, 19). The use of these compounds is prone to causing off-target
effects, and therefore independent approaches, such as genetic
manipulation of lipid biosynthetic pathways, are indispensable to
extend these observations. Evidence for the role of lipid rafts
in viral glycoprotein transport comes mainly from studies that
target cholesterol levels. Even though SM is a key component of
lipid rafts, its exact role in protein transport and viral biogenesis
remains largely unknown. Here we describe the consequences
of a defect in SM synthesis caused by inactivation of the SM synthase-1 (SGMS1) gene, as brought about by insertional mutagenesis (20) and pharmacological blockade (21). SGMS1-null
cells and their wild-type counterparts are susceptible to infection
with influenza and show similar patterns of glycoprotein maturation in transit from the ER through the Golgi apparatus, but
SGMS1-deficient cells and myriocin-treated Maldin–Darby canine kidney (MDCK) cells show a major delay in maturation and
release of new virus particles.
Author contributions: F.G.T., S.S., J.A., C.P.G., M.H., P.S., and H.L.P. designed research; F.G.T.,
S.S., J.A., C.P.G., and M.H. performed research; F.G.T., S.S., J.A., C.P.G., and M.H. contributed
new reagents/analytic tools; F.G.T., S.S., J.A., C.P.G., M.H., P.S., and H.L.P. analyzed data; and
F.G.T., S.S., J.A., C.P.G., P.S., and H.L.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: ploegh@wi.mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1219909110/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1219909110
Characterization of SGMS1-Deficient KBM7 Cells. SGMS1 catalyzes
the transfer of phosphorylcholine from phosphatidylcholine (PC)
onto ceramide to produce SM (Fig. 1A); SGMS1 is the major
SM synthase in mammalian cells (22). To examine the role of
SM in influenza virus infection, we used SGMS1GT (SGMS1gene trap), a cell line isolated in a screen in a human haploid cell
line selected for resistance to cytolethal distending toxins produced by various pathogenic bacteria (20, 23). SGMS1 transcripts are present in the parental cell line KBM7 but are absent
from SGMS1GT cells, as shown by RT-PCR (Fig. 1B). We determined the ability of these cells to synthesize SM by metabolic
labeling of KBM7 and SGMS1GT cells with the SM precursor Nmethyl-[14C]-choline. We extracted total lipids followed by TLC
and autoradiography. SGMS1GT cells showed reduced SM synthase activity (∼15%) compared with the KBM7 cells (Fig. 1 C
and D). The residual activity is due to the second enzyme,
SGMS2, which accounts for 10–20% of SM synthase activity in
mammalian cells (24). We found no differences in the synthesis
of [14C]-PC between SGMS1GT and KBM7 cells (Fig. 1 C
and D).
SGMS1-Deficient KBM7 Cells Have Significantly Reduced Levels of SM.
To assess the contribution of SGMS1 to sphingolipid content at
steady state, we determined the lipid composition of SGMS1GT
and KBM7 cells using liquid chromatography–tandem mass spectrometry. We find that SGMS1GT cells have ∼20% of total cellular
myriocin
ER
palmitoyl-CoA
serine
SPT
CoA+CO2
SGMS2
ceramide
[14C]-PC
[14C]-SM
10
F
5
0
SM
Cer
Glc
Cer
KBM7
GT
SGMS1
80
60
40
20
SM
Lac
Cer
% mol (of total lipids)
% mol (of total lipids)
KBM7
SGMS1GT
100
0
Origin
E
ceramide
% relative to control
D
GAPDH
15
Phosphatidylglycerol
CERT
KBM7 SGMS1GT
SGMS1
C
Diacylglycerol
SGMS1
3-ketodihydrosphingosine
B
Golgi/PM
sphingomyelin
PC
80
60
KBM7
SGMS1GT
40
G
20
0
PC
PE
PS
PI
% mol (of control)
A
KBM7
SGMS1GT
100
50
0
Phos
Chol
Fig. 1. SGMS1GT cells have reduced levels of SM. (A) Schematic representation of the SM biosynthesis pathway in mammalian cells. SGMS1-deficient
KBM7 cells and myriocin, an SPT inhibitor (marked in box) show a block in
sphingolipid biosynthesis. (B) RT-PCR analysis showing the absence of the
SGMS1 transcript in SGMS1GT cells. (C) Autoradiogram of a TLC separation of
lipid extracts of KBM7 and SGMS1GT cells metabolically labeled with the SM
precursor [14C]-choline. (D) Quantification of the [14C]-SM and [14C]-PC signals from [14C]-choline labeling experiment in C. (E and F) Total lipids were
extracted from KBM7 and SGMS1GT cells and subjected to MS/MS. Levels of
sphingolipids (E) and glycerophospholipids (F) are expressed as mole percent
of total lipid analyzed. (G) Total phosphate and cholesterol levels of KBM7
and SGMS1GT cells were determined and presented as mole percent relative
to the control. Error bars represent SD of three (D) or two (E–G) independent
experiments. Cer, ceramide; PC, phosphatidylcholine; PS, phosphatidylserine;
PI, phosphatidylinositol.
Tafesse et al.
SM content compared with KBM7 (Fig. 1E), which corroborates
our metabolic labeling experiments (Fig. 1 C and D). Lipid
analysis of cells that were cultured for more than 2 wk in delipidated media showed a similar reduction of SM level. Hence,
the remaining SM produced in SGMS1GT cells must result from
the activity of SM synthase-2 (SGMS2). The decrease in SM
levels in SGMS1GT cells was accompanied by a twofold increase
of its precursor, ceramide (Fig. 1E). Ceramide is also a precursor
for the synthesis of lactosylceramide (LacCer) and glucosylceramide (GlcCer). In SGMS1GT cells the level of LacCer increased
fourfold, whereas the level of GlcCer increased twofold compared with KBM7 cells (Fig. 1E). The species of SM present in
KBM7 and SGMS1-deficient cells were indistinguishable (Fig.
S1). Relative to KBM7 cells, PC levels were higher, whereas
phosphatidylethanolamine (PE) levels were lower in SGMS1GT
cells (Fig. 1F). Cholesterol content and overall phosphate levels
remain unchanged (Fig. 1G). Combined, our data show that
SGMS1GT cells have significantly reduced levels of SM compared with the parental KBM7 line, consistent with the known
defect in SM synthesis.
SGMS1-Deficient Cells Are Defective in Influenza Virus Production. To
examine the role of SM in the context of virus infection, we
first assessed the extent to which SGMS1GT cells support an
infection with influenza virus. We infected SGMS1GT and KBM7
cells at a multiplicity of infection (MOI) of 0.1. At 24 h after
infection, cells were pulse-labeled with [35S]-methionine/cysteine.
We analyzed total cell lysates by SDS/PAGE and autoradiography. We detected slightly more newly synthesized host proteins
in SGMS1GT compared with KBM7 cells (Fig. 2A). This suggests
a possible defect in viral production and in reduction of host
protein synthesis in SGMS1GT cells, which therefore may be less
able to sustain virus replication. The major viral proteins detected
correspond to nucleoprotein (NP) and M1 (Fig. 2A). This led us
to examine virus production by SGMS1GT cells. To this end,
KBM7 and SGMS1GT cells were infected at an MOI of 0.1, and at
24 h after infection the media was collected and a fluorescent
focus assay performed. SGMS1GT cells produce less than half the
amount of virus than do KBM7 cells (Fig. 2B).
SGMS1-Deficient Cells Bind Influenza Virus in Amounts Similar to
Those Bound by KBM7 Cells. Because SGMS1GT cells show im-
paired production of influenza virus, we explored the step at
which SM is required in the life cycle of the virus. To compare
the extent of virus binding to KBM7 and SGMS1GT cells, we
used a modified strain of influenza virus (HA-Srt), labeled sitespecifically in its HA1 moiety with Alexa 647 (25). SGMS1GT
and KBM7 cells were incubated with an equal amount of Alexa
647-labeled virus and/or cholera toxin (CTx) for 30 min on ice to
prevent endocytosis and analyzed by cytofluorimetry or confocal
microscopy. SGMS1GT and KBM7 cells bound similar amounts
of labeled virus (Fig. 2 C and E), indicating that SM is not essential for adsorption of influenza virus. Additionally, SGMS1GT
cells and KBM7 bound similar amounts of labeled CTx, particularly at lower concentrations of CTx (Fig. 3 D and E).
Influenza Virus HA Transport from the ER to the Golgi Is Not Affected
in SGMS1-Deficient KBM7 Cells. Because initial binding of influenza
virus to KBM7 and SGMS1GT cells was similar, we next investigated the trafficking of viral glycoproteins through the secretory pathway. In yeast, sphingolipid synthesis is critical for
ER-to-Golgi transport of glycosylphosphatidylinisotol (GPI)anchored proteins such as Gas1p (26), but the role of sphingolipids in glycoprotein trafficking in mammalian cells is less clear.
We therefore examined the synthesis and maturation of influenza HA in KBM7 and SGMS1GT cells. The HA precursor
(HA0) trimerizes and traffics through the secretory pathway, en
route acquiring complex-type N-linked glycans in the Golgi
PNAS | April 16, 2013 | vol. 110 | no. 16 | 6407
CELL BIOLOGY
Results
4000
3000
75
50
NP
37
1000
M1
25
2000
0
kDa
C
SGMS1GT
Mock
0.5 MOI
1 MOI
% max
KBM7
KBM7 SGMS1GT
FL4
D
KBM7
FL4
SGMS1GT
% max
0.2nM
FL4
E
1nM
10nM
FL4
Alexa488-CTx
FL4
Merge
SGMS1GT
KBM7
Alexa647-HA-Srt
FL4
SLC25A2GT
Fig. 2. SGMS1GT cells are defective in influenza viral particles production.
(A) Equal numbers of KBM7 and SGMS1GT cells were infected with influenza
virus (A/WSN/33) at an MOI of 0.1. At 24 h after infection, cells were pulselabeled with [35S]-methionine/cysteine for 30 min and total cell lysates were
analyzed by SDS/PAGE and autoradiography. (B) Cells were infected as described in A, and viral titers from the supernatant were measured by
a fluorescent focus assay on MDCK cells. (C) Cells were incubated at different
MOIs with Alexa 647-labeled influenza virus for 30 min on ice, followed by
cytofluorimetry. (D) Flow cytometry of Alexa 647-positive cells upon exposure of KBM7, SGMS1GT, and SLC25A2GT with CTx-Alexa647 for 15 min on
ice. SLC25A2GT cells are defective in GM1 biosynthesis and hence CTx is unable to bind to these cells. (E) Confocal images of KBM7 and SGMS1GT cells
incubated with Alexa 647-labeled influenza virus and Alexa 488-CTx for 30
min on ice. FFU, focus-forming unit.
(27, 28). Transport of HA from the ER to the Golgi apparatus
proceeds normally in SGMS1GT cells, as assessed by the rate and
extent of acquisition of Endoglycosidase H (Endo H) resistance
(Fig. 3 A and B). Similarly, we found no measurable delay in
trafficking of HA from the ER to the Golgi in MDCK cells
treated with myriocin, a specific inhibitor of serine palmitoyltransferase (see below and Fig. 4B).
Influenza Virus HA and NA Transport from the Golgi to Cell Surface Is
Affected in Cells Defective of SM Biosynthesis. SGMS1 is localized
in the Golgi, where it catalyzes the production of SM on the
luminal side of the trans-Golgi network (TGN). On the basis of
our analysis of SGMS1GT cells, we hypothesized that a lack of
SM in the Golgi compartment might affect membrane organization and hence impede trafficking of HA. To explore this, we
infected SGMS1GT and KBM7 cells with influenza virus. We
monitored arrival of metabolically labeled HA at the cell surface
by trypsin treatment, which cleaves surface-disposed but not
6408 | www.pnas.org/cgi/doi/10.1073/pnas.1219909110
intracellular HA0 to yield two fragments, HAl and HA2 (29).
Transport of HA from the Golgi to the cell surface was considerably delayed in SGMS1GT cells (Fig. 3 C and D). At 30 min of
chase, most of HA in KBM7 cells had reached the cell surface,
whereas only ∼5% arrived in SGMS1GT cells. However, at 60 min
of chase, the difference was less pronounced, with ∼70% of HA
arriving at the cell surface of SGMS1GT cells (Fig. 3 C and D).
We next determined whether SGMS1GT cells have a more
general defect in protein transport. We monitored the level of
secreted proteins in KBM7 and SGMS1GT cells. KBM7
and SGMS1GT cells were labeled for 30 min with [35S]methionine/cysteine and chased for different times. Proteins
secreted into the media were analyzed by SDS/PAGE and autoradiography. As a negative control, we incubated cells at 4 °C to
block secretion. There was no difference in the levels of secreted
proteins when comparing SGMS1GT and KBM7 cells (Fig. S2B).
In parallel, we monitored the transport of class I MHC proteins—
type I membrane glycoproteins that traffic through the constitutive secretory pathway to reach the cell surface (30). SGMS1GT
cells show no significant difference in the transport of class I
MHC (Fig. 3 E and F), as assessed by rate and extent of acquisition of Endo H resistance.
Myriocin is a potent inhibitor of serine palmitoyltransferase
(SPT), an enzyme that catalyzes the first and rate-limiting step
of de novo sphingolipid biosynthesis (21). Consequently the
levels of ceramide—the substrate for SGMS1 and SGMS2—are
expected to be significantly lower in myriocin-treated cells. We
treated MDCK cells with 100 nM myriocin for 72 h before virus
infection. We determined the extent of inhibition of SM synthesis by metabolic labeling with [14C]-serine or [14C]-choline of
myriocin-treated and control MDCK cells and visualized the
formation of [14C]-SM by TLC. Cells treated with myriocin
A
75 -
0
KBM7
30 60
50 -
SGMS1GT
0 30
60 (min)
- HA0 + gly
- HA0 - gly
- HA1
B
80
37 25 kDa
C
75 -
0
KBM7
30
60
D
- HA1
37 -
KBM7
0
30
90
SGMS1GT
0
30 90 chase (min)
MHC I + gly
MHC I - gly
30
60
time (min)
KBM7
SGMS1GT
60
40
20
0
F
0
100
80
- M1
- HA2
25 kDa
37
kDa
20
0
- HA0 - gly
50 -
E
40
- M1
- HA2
SGMS1GT
0 30 60 (min)
SGMS1GT
KBM7
60
HA (% of total)
B
HA (% of total)
SGMS1GT
Virus
+
MHC I+gly
(% of total)
150
KBM7
+
FFU/ml
A
80
60
40
20
0
0
30
time (min)
60
KBM7 GT
SGMS1
0
30
90
time (min)
Fig. 3. SGMS1GT cells are defective in trafficking of HA from the Golgi to
the cell surface but not from the ER to Golgi. (A and C) KBM7 and SGMS1GT
cells were infected at an MOI of 0.5. At 5 h after infection, cells were pulselabeled with [35S]-methionine/cysteine for 10 min and chased for the indicated time points in the presence of trypsin in the chase medium. HA
molecules were recovered by immunoprecipitation using anti-HA serum,
subjected to Endo H (A) or PNGase F (C) treatment, and analyzed by SDS/
PAGE and autoradiography. (E) KBM7 and SGMS1GT cells were pulse-labeled
with [35S]-methionine/cysteine for 10 min and chased for different time
points. Cells were lysed and class I MHC molecules were recovered using
W6/32 antibody, treated with Endo H, and analyzed by SDS/PAGE and
autoradiography. (B, D, and F ) Quantification of experiments performed
as described in A, C, and E, respectively. Error bars, SD of three independent experiments.
Tafesse et al.
[14C]-Choline
+
[14C]-Serine
+
C
[14C]-GC
[14C]-PC
[14C]-SM
75 -
- myriocin
0
1
2
50 -
+ myriocin
0
1
2 (h)
- HA0
(S)
75 -
H
HA0
((Golgi)
HA0
(ER)
50 -
Origin
D
B
+ myriocin
- myriocin
- myriocin
+ myriocin
0 30 60 90 0 30 60 90 (min)
0 30 60 90 0 30 60 90 (min)
75 - HA0
H + gly 75 50 -
- HA0
H - gly
50 -H
HA1
37 -
37 -
E
-
- myriocin
4 5 6 7
50 50 37 kDa
+ myriocin
4 5 6 7 p.i. (h)
- NA (S)
F
- NA (total)
- GAPDH
M
M1
25 kDa
cell surface NA
(% of total)
*
HA2
H
25 kDa
- NP
- HA1
H
HA2
- myriocin
+ myriocin
40
20
0
4
5
6
time (p.i., h)
7
Fig. 4. Myriocin-treated MDCK cells are defective in cell-surface display but
not ER to Golgi transport of influenza HA and NA. (A) MDCK cells treated
with myriocin for 3 d were labeled with [14C]-choline or [14C]-serine for 4 h
and lipids were extracted and analyzed by TLC. (B) MDCK cells treated with
myriocin as described in A were infected with influenza virus at an MOI of
0.5. At 5 h after infection, cells were pulse-labeled with [35S]-methionine/
cysteine for 10 min and chased for the indicated time points in the presence
of trypsin added to the chase medium. Cells were lysed and HA molecules
were recovered using anti-HA serum, subjected to Endo H treatment, and
analyzed by SDS/PAGE and autoradiography. (C) MDCK cells treated with
myriocin as described in A were infected at an MOI of 0.5 with HA-Srt virus,
and at 5 h after infection cells were pulse-chased as described in B. At the
indicated time points, surface-accessible HA-Srt was labeled with GGGKbiotin in the presence of sortase A for 1 h on ice. Cells were lysed, and biotinlabeled HA was recovered by affinity adsorption on NeutrAvidin agarose
beads. (D) MDCK cells treated with myriocin and infected as described in C
were pulse-labeled with [35S]-methionine/cysteine for 10 min. At the indicated time points the supernatants were collected and incubated with
chicken erythrocytes to recover radiolabeled virus released into the media.
Samples were analyzed as described in B. (E) Myriocin-treated MDCK cells
were infected with NA-Srt virus and at the indicated time points after infection, cells were labeled with GGGK-biotin in the presence of sortase A for
1 h on ice. Cells were lysed, and biotinylated NA (= surface-exposed) was
detected by immunoblotting using streptavidin-HRP (E, Upper), and total NA
(= surface + intracellular) was detected using anti-HA epitope tag-HRP (E,
Middle). (F) Quantification of experiments performed as described in E. Error
bars, SD of three independent experiments. *The identity of this protein
band is unknown.
showed a near-complete blockade of SM production, whereas
the biosynthesis of other phospholipids such as [14C]-PC and
[14C]-PS remained unaffected (Fig. 4A).
We monitored the arrival of HA at the cell surface by infecting
MDCK cells with a “sortaggable” influenza virus (HA-Srt or NASrt), which allows site-specific incorporation of label in surfacedisplayed HA or NA (25). We infected myriocin-treated and
control MDCK cells with HA-Srt and at 5 h after infection labeled them with [35S]-methionine/cysteine. At different chase
times, cells were placed on ice and subjected to sortagging with
biotin (25). We recovered biotinylated (= surface-disposed) HA
using NeutrAvidin beads. We detected no biotinylated HA at the
cell surface in myriocin-treated MDCK cells even at the 90-min
chase point, whereas the majority of the biotinylated HA had
reached the cell surface at 30 min chase point in control cells
(Fig. 4C). The effects of SM depletion on surface display of HA
are more severe than those recorded for methyl-β-cyclodextrin–
mediated reduction of cholesterol levels (31).
Tafesse et al.
We also examined trafficking of NA in myriocin-treated and
untreated MDCK cells infected with NA-Srt virus (25), which
encodes an NA that carries an HA epitope tag distal to the
sortase recognition sequence. Surface-accessible NA was labeled
with GGGK-biotin in the presence of sortase A enzyme for 1 h
on ice (25). We used this approach because the abundance of
NA is far less than that of HA, complicating its detection by
metabolic labeling (32). Biotinylated (= surface-disposed) NA
was detected in total cell lysate in immunoblots using streptavidin-HRP (Fig. 4E, Upper). Total NA levels (= surface + intracellular pools) were measured using an HRP-conjugated antiHA epitope antibody, which does not recognize the HA of the
A/WSN/33 virus (Fig. 4E, Middle). We observed a significant
delay in the arrival of NA at the cell surface in myriocin-treated
cells (Fig. 4 E and F). Sphingolipid biosynthesis is thus required
for surface display of both HA and NA.
To determine the effect of myriocin treatment on the level of
influenza virus production we infected myriocin-treated and
control MDCK cells with the virus and performed pulse-chase
experiment at 5 h after infection. At different time points during
the chase the supernatants were collected and incubated with
chicken erythrocytes to recover radiolabeled virus released into
the media. Consistent with the SGMS1GT results, we found a
strongly reduced level of viral proteins in the supernatants of
myriocin-treated MDCK cells (Fig. 4D), indicating that intact
sphingolipid biosynthesis is required for the efficient production
of influenza virus. Collectively, our data show that proper
sphingolipid biosynthesis makes a key contribution to surface
display of flu glycoproteins and hence virus particle formation
and release.
We then determined whether SM depletion disrupts trafficking of endogenous lipid raft-associated proteins by monitoring
the rate of the recovery of Thy1, a glycosylphosphatidyl-anchored
membrane glycoprotein, on the cell surface after its removal by
phosphatidylinositol-specific phospholipase C (PI-PLC) digestion.
Myriocin-treated and control EL4 cells were treated with PI-PLC
for 1 h at room temperature. After extensive washing to remove
traces of PI-PLC, cells were returned to 37 °C. At different time
points, the cells were costained with anti-Thy1-PE and anti-class I
MHC-FITC and analyzed by FACS (Fig S2A). We found only
slightly less Thy1 on the cell surface of myriocin-treated cells
at 3 h incubation point compared with untreated EL4 cells
(Fig S2A, Upper) and no difference with respect to class I MHC
levels (Fig S2A, Lower).
Influenza Virus HA and NA Show Increased Triton X-100 Solubility
upon Blockade of SM Biosynthesis. Influenza virus HA and NA
are known to associate with lipid rafts (17, 19, 33), but the role of
SM in this process remains largely unknown. Lipid rafts and their
associated proteins show impaired solubility in nonionic detergents such as Triton X-100 (TX-100) or Nonidet P-40 at 4 °C
(34). Because transport of HA from the TGN to the cell surface
is impaired in SGMS1-deficient cells, we examined the solubility
of HA in cold nonionic detergent. For this purpose, virusinfected KBM7 and SGMS1GT cells were pulse-labeled with
[35S]-methionine/cysteine for 10 min, chased for different time
points, and subjected to TX-100 extraction on ice. At the 0-h
time point HA resides in the ER, whereas at 2 h the majority of
HA localizes to the Golgi/post-Golgi compartment, as assessed
by acquisition of Endo H resistance. This helped identify the
compartment where SM is necessary for the interaction of HA
with lipid rafts. At the 0-h time point, almost all HA was found in
the soluble fraction, both in KBM7 and SGMS1GT cells. However, at the 2-h time point HA remained soluble in cold TX-100
in SGMS1GT cells, whereas in KBM7 cells a far greater fraction
was detergent-insoluble (Fig. 5C). Treatment of MDCK cells
with myriocin also yielded an increased fraction of TX-100 soluble HA (Fig. 5 A and B). Similarly, to measure cold detergent
PNAS | April 16, 2013 | vol. 110 | no. 16 | 6409
CELL BIOLOGY
Amyr
A
0h
Myr
P
+
S
D
2h
P
S
P
P
P
S
6h
+
S
P
S -
P
+
S
P
S
NA -
40
30
20
10
0
0h
2h
C 40
E
KBM7
SGMS1GT
30
20
80
- Myr
+ Myr
60
40
10
0
100
% insoluble NA
- Myr
+ Myr
% insoluble HA
50
% insoluble HA
-
+
S
HA -
B
2h
20
0h
2h
0
2h
6h
Fig. 5. Influenza virus HA and NA retains TX-100 solubility upon blockade
of sphingolipid biosynthesis. (A) Myriocin-treated and control MDCK cells
were infected with influenza virus, and at 5 h after infection, cells were
pulse-labeled with [35S]-methionine/cysteine for 10 min, chased for indicated
time points, and subjected to cold 1% TX-100 extraction. HA molecules from
the soluble (S) and insoluble (P) fractions were recovered using anti-HA serum, analyzed by SDS/PAGE, and autoradiography. (B) Quantification of
three independent experiments performed as described in A or using KBM7
and SGMS1GT cells (C). (D) Myriocin-treated and control MDCK cells were
infected with NA-Srt virus that carries the HA-epitope tag in addition to
a sortase recognition motif. At the indicated time after infection, cells were
subjected to cold T-X100 extraction. NA molecules from the soluble and
insoluble fractions were detected by Western blotting using anti-HA epitope
tag-HRP. (E) Quantification of three independent experiments performed as
described in D.
extractability of NA, we probed for NA at various time intervals
after infection. At 2 h after infection we found NA to be soluble
in cold TX-100 both in myriocin treated and untreated MDCK
cells, presumably still residing in the early compartments of the
secretory pathway. At 6 h after infection, a large fraction of NA
in mock-treated MDCK cells becomes insoluble in cold TX-100,
whereas in myriocin-treated cells the bulk of NA remains soluble
(Fig. 5 D and E).
Discussion
The assembly of influenza virus particles requires not only viral
components themselves but also many host factors essential for
glycoprotein maturation, (glyco)lipid synthesis, and vesicular
transport. Genome-wide RNAi screens conducted for influenza
virus and performed in different cell types have uncovered many
host proteins proposed to contribute to virus assembly, but
enzymes involved in lipid synthesis have not been identified in
such screens (5–7). The SM synthase-1 null cell line (SGMS1GT)
used in this study was isolated from the collection of mutants
resistant to intoxication by cytolethal distending toxins (20, 23).
SGMS1GT cells show a starkly reduced level of SM (15% of wild
type) with the concomitant expected increase in its precursors—
ceramide and phosphatidylcholine—and in other lipids that use
ceramides as a precursor as well. Because of the pronounced
preference of influenza virus HA for membrane domains
enriched in cholesterol and glycolipids (35), we explored the role
of SM content as a parameter that contributes to assembly of
new influenza virions. Evidence for the role of lipid rafts in HA
and NA trafficking mostly relies on cholesterol depletion by
methyl-β-cyclodextrin (18, 31, 36). Although SM is believed to be
a major component of rafts, scant evidence exists for a direct role
of SM on HA/NA intracellular transport. In addition, contradictory reports on the effect of cholesterol depletion on influenza
virus budding indicate that this question is far from solved (37).
To this end we used SGMS1GT cells and myriocin, a compound
that blocks SM synthesis. Interference with SM synthesis does
not affect binding of influenza virus to the cell surface, and only
minimally affects the ability of the virus to infect the cell.
6410 | www.pnas.org/cgi/doi/10.1073/pnas.1219909110
HA0 trimerizes in the ER and assumes its native fold as it
prepares to exit for the Golgi, a protracted process that involves
imposition of the complex disulfide-bonding pattern to stabilize
the native trimer (38). How a lipid annulus of defined composition is organized around the membrane anchors of HA and, by
extension, NA is not known, but interactions might be through
palmitoylation (39) and/or through the polypeptide segments
interposed between the transmembrane anchors and the luminal
portions with well-defined tertiary structure (18, 36). We show
that compromised SM synthesis does not interfere with maturation of the N-linked glycans on HA, indicating that the transport of HA from the ER to the Golgi is not dependent on SM.
The block in maturation of viral particles in SGMS1GT cells and
myriocin-treated cells therefore must occur at or beyond the
trans-Golgi network.
Consistent with this notion, we found that surface display of
viral glycoproteins is affected when sphingolipid biosynthesis is
compromised. We established this using three independent criteria: access of cell surface-exposed HA or NA to trypsin and
sortase, as well as inhibition of release of virus particles. SM thus
contributes to protein sorting from the TGN to the cell surface.
The formation of protein carrier vesicles that bud from the TGN
to deliver their cargo to the cell surface requires SM (40), and
perturbation of SM biosynthesis might therefore affect cargo
sorting. SM has affinity for cholesterol and forms lipid microdomains with distinct protein composition (41). Depletion of
cholesterol through application of compounds such as methylβ-cyclodextrin and lovastatin/mevalonate affect trafficking of raftassociated proteins, including GPI-anchored and some transmembrane proteins, such as influenza HA (42). As shown here,
removal of SM is sufficient to cause a viral glycoprotein trafficking
defect in its own right. Although SGMS1GT cells showed increased levels of ceramide and the glycosphingolipids LacCer and
GlcCer, known to form lipid microdomains (43), they do not
compensate for the reduction of SM as a requirement for proper
trafficking of HA and NA, suggesting the possible existence of
distinct classes of lipid microdomains with distinct biological
roles. Overall, our data demonstrate how influenza virus and
possibly other enveloped viruses exploit host factors such as cellular lipids during their life cycle. Interference with these pathways may provide new means of blunting the pathological effects
caused by enveloped viruses.
Methods
Virus Propagation, Infection, and Fluorescent Focus Assay. MDCK cells were
cultured in DMEM supplemented with 10% serum, whereas KBM7 cells were
grown in Iscove’s modified Dulbecco’s media (IMDM) with 10% serum. Influenza virus A/WSN/33 was propagated in MDCK cells. Infection of KBM7,
SGMS1GT, and MDCK cells was performed as follows: cells were washed twice
with PBS and subsequently incubated with virus diluted in PBS/0.2%/1 μg/mL
of trypsin at room temperature for 30 min at the given MOI. Cells were then
washed once in PBS to remove unbound virus and then placed into fresh
DMEM supplemented with 0.2% BSA and 1 μg/mL of trypsin and incubated
at 37 °C, 5% CO2. For quantifying virus titer, MDCK cells were infected with
serial dilutions of supernatant. After infection, cells were washed and then
incubated in 0.8% agar/MEM overlay. After 2 d, the overlay was removed,
cells were fixed, and virus foci were detected via immuno-fluorescence using
an NP-FITC antibody.
Myriocin Treatment of MDCK Cells. MDCK cells were either mock treated (only
DMSO) or treated with 0.5 μg/mL myriocin (in DMSO; Sigma Aldrich) for 72 h,
after which cells were infected with influenza virus. Reduced levels of SM in
myriocin-treated cells, was verified by lipid analysis as described. Biosynthetic labeling of viral proteins was unaffected in myriocin-treated cells
as observed by total radiolabel incorporated.
Pulse Chase Experiments, Sortase Labeling, and Glycosidase Digestion. MDCK
cells were grown in 10-cm culture dishes and infected with either wild-type
influenza virus WSN or engineered HA-Srt or NA-Srt virus (25) at an MOI of
0.5 for 4 h. In addition to sortase recognition sequence (LPETG), NA-Srt
Tafesse et al.
sample buffer and resolved by SDS/PAGE. Samples were visualized with
autoradiography using DMSO/PPO (2,5-diphenyloxazole) and exposure to
Kodak XAR-5 film. Densitometric quantification of radiograms and immunoblots were performed using Photoshop/Image J software.
carries an HA epitope tag (25). KBM7 cells (wild type and SGMS1-deficient)
cells were grown in suspension in 24-well plates to a density of 2 × 106 cells
per well before infection. Cells were harvested and resuspended in methionine- and cysteine-free DMEM and starved for 45 min at 37 °C followed by
a 10-min pulse labeling with [35S]-methionine/cysteine at 0.77 mCi/mL and
chase in complete media for 0, 30, and 60 min. Where indicated, 1 μg/mL
trypsin was added in the chase media. To probe for surface exposed HA at
indicated time points during the chase, cell-surface HA molecules were labeled with 250 μM GGGK-biotin and 100 μM SrtA for 1 h at 4 °C. To measure
total intracellular HA, cell pellets were collected, lysed in Tris buffer [150 mM
NaCl, 5 mM MgCl2, 25 mM Tris (pH 7.4)] containing 0.5% Nonidet P-40
followed by immunoprecipitation with influenza virus anti-HA serum. For
MDCK cells infected with NA-Srt virus, at indicated times after infection, cells
were incubated for 1 h with 100 μM SrtA and 250 μM GGGK-biotin. Cells
were lysed in buffer containing SDS, and biotinylated NA (= surfacedisposed) was detected by Western blotting from the total lysate using
streptavidin-HRP. The total NA (= surface + intracellular) was determined using
anti-HA epitope tag-HRP (3F10). This antibody (3E10) does not recognize the
HA of A/WSN/33.
For class I MHC pulse chase experiment, 10 × 106 KBM7 and SGMS1GT cells
were labeled with [35S]-methionine/cysteine for 10 min and chased as indicated in the figure legend. At different time points during the chase cells
were collected, washed once with cold PBS, and lysed in Tris buffer [150 mM
NaCl, 5 mM MgCl2, 25 mM Tris (pH 7.4)] containing 0.5% Nonidet P-40. The
lysates were precleared with immobilized protein G beads for 3 h, and class I
MHC molecules were recovered using the monoclonal antibody (W6/32) to
class I MHC. For all experiments, where indicated, immunoprecipitated
samples were subjected to Endo H or PNGase F treatment according to the
manufacturer’s instructions. Immunoprecipitates were eluted with SDS
TX-100 Extraction. TX-100 extraction was performed according to refs. 13 and
35. For HA extraction, virus-infected cells were pulse-labeled with [35S]methionine/cysteine at 0.77 mCi/mL for 10 min and chased for different
time points. Cells were harvested, washed once with cold PBS, and
resuspended in 0.5 mL of ice-cold Hepes-plus buffer [25 mM Hepes (pH
7.4), 150 mM NaCl, 1 mM EDTA, and a protease inhibitor mixture (Roche)].
Extraction was initiated by addition of 0.5 mL 2% (wt/vol) TX-100 in Hepes-plus
buffer (1% TX-100 final concentration) for 30 min on ice. To separate the soluble from the insoluble fraction, cells were centrifuged at 12,000 × g in an
Eppendorf microfuge for 20 min at 4 °C. The pellet fraction was solubilized in
a Hepes-plus buffer containing 1% digitonin with vigorous vortexing, and HA
was immunoprecipitated from both fractions using anti-HA serum and analyzed by SDS/PAGE and autoradiography. To determine the detergent solubility
of NA protein, cells were infected with NA-Srt virus (25). At different times after
infection, cells were harvested and TX-100 extraction was done as described for
HA. The pellet fraction of NA was solubilized in SDS sample buffer, and the
amount of NA in soluble and insoluble fractions was detected by Western
blotting. The insoluble fraction was calculated as the percentage relative to the
total fraction (soluble and insoluble).
1. Shaw ML (2011) The host interactome of influenza virus presents new potential targets for antiviral drugs. Rev Med Virol 21(6):358–369.
2. Meliopoulos VA, et al. (2012) Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens. FASEB J 26(4):1372–1386.
3. Lee SM, Yen HL (2012) Targeting the host or the virus: Current and novel concepts for
antiviral approaches against influenza virus infection. Antiviral Res 96(3):391–404.
4. Müller KH, et al. (2012) Emerging cellular targets for influenza antiviral agents.
Trends Pharmacol Sci 33(2):89–99.
5. Brass AL, et al. (2009) The IFITM proteins mediate cellular resistance to influenza A
H1N1 virus, West Nile virus, and dengue virus. Cell 139(7):1243–1254.
6. König R, et al. (2010) Human host factors required for influenza virus replication.
Nature 463(7282):813–817.
7. Hao L, et al. (2008) Drosophila RNAi screen identifies host genes important for influenza virus replication. Nature 454(7206):890–893.
8. Gerl MJ, et al. (2012) Quantitative analysis of the lipidomes of the influenza virus
envelope and MDCK cell apical membrane. J Cell Biol 196(2):213–221.
9. Kalvodova L, et al. (2009) The lipidomes of vesicular stomatitis virus, semliki forest
virus, and the host plasma membrane analyzed by quantitative shotgun mass spectrometry. J Virol 83(16):7996–8003.
10. Enami M, Enami K (1996) Influenza virus hemagglutinin and neuraminidase glycoproteins stimulate the membrane association of the matrix protein. J Virol 70(10):
6653–6657.
11. Gamblin SJ, Skehel JJ (2010) Influenza hemagglutinin and neuraminidase membrane
glycoproteins. J Biol Chem 285(37):28403–28409.
12. Rossman JS, Jing X, Leser GP, Lamb RA (2010) Influenza virus M2 protein mediates
ESCRT-independent membrane scission. Cell 142(6):902–913.
13. Skibbens JE, Roth MG, Matlin KS (1989) Differential extractability of influenza virus
hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts. J Cell Biol 108(3):821–832.
14. Simons K, Gerl MJ (2010) Revitalizing membrane rafts: New tools and insights. Nat
Rev Mol Cell Biol 11(10):688–699.
15. Munro S (2003) Lipid rafts: Elusive or illusive? Cell 115(4):377–388.
16. Lingwood D, Kaiser HJ, Levental I, Simons K (2009) Lipid rafts as functional heterogeneity in cell membranes. Biochem Soc Trans 37(Pt 5):955–960.
17. Simons K, Ikonen E (1997) Functional rafts in cell membranes. Nature 387(6633):
569–572.
18. Takeda M, Leser GP, Russell CJ, Lamb RA (2003) Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion. Proc Natl Acad Sci USA
100(25):14610–14617.
19. Scheiffele P, Roth MG, Simons K (1997) Interaction of influenza virus haemagglutinin
with sphingolipid-cholesterol membrane domains via its transmembrane domain.
EMBO J 16(18):5501–5508.
20. Carette JE, et al. (2009) Haploid genetic screens in human cells identify host factors
used by pathogens. Science 326(5957):1231–1235.
21. Hojjati MR, et al. (2005) Effect of myriocin on plasma sphingolipid metabolism and
atherosclerosis in apoE-deficient mice. J Biol Chem 280(11):10284–10289.
22. Tafesse FG, Ternes P, Holthuis JC (2006) The multigenic sphingomyelin synthase
family. J Biol Chem 281(40):29421–29425.
23. Carette JE, et al. (2011) Global gene disruption in human cells to assign genes to
phenotypes by deep sequencing. Nat Biotechnol 29(6):542–546.
24. Tafesse FG, et al. (2007) Both sphingomyelin synthases SMS1 and SMS2 are required
for sphingomyelin homeostasis and growth in human HeLa cells. J Biol Chem 282(24):
17537–17547.
25. Popp MW, Karssemeijer RA, Ploegh HL (2012) Chemoenzymatic site-specific labeling
of influenza glycoproteins as a tool to observe virus budding in real time. PLoS
Pathog 8(3):e1002604.
26. Bagnat M, Keränen S, Shevchenko A, Shevchenko A, Simons K (2000) Lipid rafts
function in biosynthetic delivery of proteins to the cell surface in yeast. Proc Natl Acad
Sci USA 97(7):3254–3259.
27. Rossman JS, Lamb RA (2011) Influenza virus assembly and budding. Virology 411(2):
229–236.
28. Nayak DP, Hui EK, Barman S (2004) Assembly and budding of influenza virus. Virus
Res 106(2):147–165.
29. Wiley DC, Skehel JJ (1987) The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem 56:365–394.
30. Wiertz EJ, et al. (1996) The human cytomegalovirus US11 gene product dislocates
MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell 84(5):
769–779.
31. Keller P, Simons K (1998) Cholesterol is required for surface transport of influenza
virus hemagglutinin. J Cell Biol 140(6):1357–1367.
32. Shaw ML, Stone KL, Colangelo CM, Gulcicek EE, Palese P (2008) Cellular proteins in
influenza virus particles. PLoS Pathog 4(6):e1000085.
33. Zhang J, Pekosz A, Lamb RA (2000) Influenza virus assembly and lipid raft microdomains: A role for the cytoplasmic tails of the spike glycoproteins. J Virol 74(10):
4634–4644.
34. Lingwood D, Simons K (2007) Detergent resistance as a tool in membrane research.
Nat Protoc 2(9):2159–2165.
35. Fiedler K, Kobayashi T, Kurzchalia TV, Simons K (1993) Glycosphingolipid-enriched,
detergent-insoluble complexes in protein sorting in epithelial cells. Biochemistry
32(25):6365–6373.
36. Barman S, Nayak DP (2000) Analysis of the transmembrane domain of influenza virus
neuraminidase, a type II transmembrane glycoprotein, for apical sorting and raft
association. J Virol 74(14):6538–6545.
37. Barman S, Nayak DP (2007) Lipid raft disruption by cholesterol depletion enhances
influenza A virus budding from MDCK cells. J Virol 81(22):12169–12178.
38. Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: The
influenza hemagglutinin. Annu Rev Biochem 69:531–569.
39. Chen BJ, Takeda M, Lamb RA (2005) Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes
differ in their ability to support assembly. J Virol 79(21):13673–13684.
40. Holthuis JC, Levine TP (2005) Lipid traffic: Floppy drives and a superhighway. Nat Rev
Mol Cell Biol 6(3):209–220.
41. Foster LJ, De Hoog CL, Mann M (2003) Unbiased quantitative proteomics of lipid
rafts reveals high specificity for signaling factors. Proc Natl Acad Sci USA 100(10):
5813–5818.
42. Mayor S, Riezman H (2004) Sorting GPI-anchored proteins. Nat Rev Mol Cell Biol 5(2):
110–120.
43. Zhang Y, Li X, Becker KA, Gulbins E (2009) Ceramide-enriched membrane domains—
structure and function. Biochim Biophys Acta 1788(1):178–183.
Tafesse et al.
PNAS | April 16, 2013 | vol. 110 | no. 16 | 6411
CELL BIOLOGY
ACKNOWLEDGMENTS. This work was supported by National Institutes of
Health R01 Awards AI033456 and AI087879. Funding has also been received
from The Netherlands Organization for Scientific Research (F.G.T.) and from
Sanofi Pasteur.
Supporting Information
Tafesse et al. 10.1073/pnas.1219909110
SI Methods
Antibodies. Antiserum specific for influenza was obtained from
mice immunized with influenza virus A/WSN/33. Antiserum
against the HA protein of influenza was obtained from transnuclear mice with B cells secreting an IgG2b specific for HA.
NeutrAvidin agarose beads were from Fisher Scientific.
Analysis of Cell Supernatants. Cells were infected with influenza
virus, pulse-labeled with [35S]-methionine/cysteine, and chased as
described above. At indicated time points, supernatant was collected, and residual cells were removed by centrifugation. For
measuring total levels of radiolabeled virus, the supernatant was
incubated with chicken erythrocytes for 1 h at 4 °C with gentle
agitation. Virus bound to erythrocytes were collected by centrifugation, washed once with PBS and lysed in SDS sample buffer,
and resolved by SDS/PAGE followed by autoradiography.
Lipid Metabolic Labeling and TLC Analysis. Approximately 2 × 10
6
KBM7 and SGMS1GT cells or MDCK cells grown in six-well dishes
were labeled with 3 μCi/mL of N-methyl-[14C]-choline (Thermo
Scientific) or 3-L-[14C]-serine (Perkin-Elmer) for 4 h in Opti-MEM
at 37 °C. Cells were washed twice in PBS and then subjected to
lipid extraction following the Bligh and Dyer method (1). The
methanol/chloroform extracts (lower face) were dried under a gentle flow of N2. Lipids were dissolved in chloroform, methanol (1:2,
vol/vol), and separated by TLC, which was developed first in acetone
and then in chloroform, methanol, 25% ammonia solution (50:25:6,
vol/vol/vol). Radiolabeled lipids were detected on a PhosphorImager (Fujifilm BAS-2500) using Image Reader BAS-2500 V1.8
(Fujifilm) and quantified using Quantity One software (Bio-Rad).
lipid molecular species and classes were quantified by liquid
chromatography-MS as detailed by Hermansson et al. (5) using
an Interchrom Lichrosphere diol-modified silica column coupled
on line to a Quattro Micro triple quadrupole mass spectrometer
(Micromass).
Flow Cytometry Analysis and Microscopy. Expression, purification,
and labeling of cholera toxin (CTx) were performed as described
in ref. 6. The FACS assays were performed with Alexa 647labeled influenza virus generated as previously described (7) or
with CTx conjugated to GGGK-Alexa 647—specifically at the C
terminus of its A1 subunit—using sortase A (6). Equal numbers
of KBM7 and SGMS1GT cells were incubated at different multiplicities of infection (MOIs) with Alexa 647-labeled virus (the
MOI was determined according to hemagglutination assay) or
CTx-Alexa 647 and propidium iodide (PI) for 15 min on ice. The
cells were washed with cold PBS supplemented with 1% BSA
and subjected to cytofluorometry immediately (FACSCalibur;
BD Biosciences). Control samples were prepared using PI only.
Only cells negative for PI staining were gated for data acquisition. FlowJo was used to analyze the data. Intensity of fluorescence was measured, and the percent maximum presented in the
overlaid histograms. For microscopy, cells were incubated with
Alexa 647-labeled influenza virus and Alexa 488-labeled CTx
subunit B (Invitrogen) on ice as described above. Images were
captured using a confocal microscope with a 63× 1.40 N.A. Plan
Apo oil objective (Carl Zeiss).
Lipid Mass Spectrometry Analysis. Lipid extracts from KBM7 and
SGMS1GT cells were prepared according to Folch et al. (2) but
without the salt. Crude lipid extracts were evaporated under N2,
dissolved in chloroform:methanol (1:2), and supplemented with
internal standards for sphingomyelin (SM), phosphatidylcholine,
phosphatidylethanolamine (PE), phosphatidylserine, phosphatidylinositol, ceramide, and GlcCer as previously described (3).
Phospholipid concentration was determined by phosphate analysis and sphingolipid concentration, and cholesterol concentrations were determined according to Tafesse et al. (4). The
Phosphatidylinositol-Specific Phospholipase C Assay. EL4 cells
grown in RMPI supplemented with 10% FCS were treated
either with myricoin or vehicle (DMSO) for 3 d. After washing
once with PBS, the cells were subjected to 1 U/mL of Bacillus
cereus phosphatidylinositol-specific phospholipase C (PIPLC) (Sigma Aldrich) digestion in Opti-MEM for 1 h at room
temperature to release glycosylphosphatidylinisotol (GPI)anchored proteins from the cell surface. Cells were washed
three times with PBS, fresh media was added, and the cells
were placed at 37 °C. At different times thereafter, cells were
collected and costained with anti-Thy1.2-PE and anti-class I
MHC-FITC on ice for 20 min and analyzed by FACS as described above.
1. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can
J Biochem Physiol 37(8):911–917.
2. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and
purification of total lipides from animal tissues. J Biol Chem 226(1):497–509.
3. Vacaru AM, et al. (2009) Sphingomyelin synthase-related protein SMSr controls
ceramide homeostasis in the ER. J Cell Biol 185(6):1013–1027.
4. Tafesse FG, et al. (2007) Both sphingomyelin synthases SMS1 and SMS2 are required for
sphingomyelin homeostasis and growth in human HeLa cells. J Biol Chem 282(24):
17537–17547.
5. Hermansson M, et al. (2005) Mass spectrometric analysis reveals changes in
phospholipid, neutral sphingolipid and sulfatide molecular species in progressive
epilepsy with mental retardation, EPMR, brain: a case study. J Neurochem 95(3):
609–617.
6. Guimaraes CP, et al. (2011) Identification of host cell factors required for intoxication
through use of modified cholera toxin. J Cell Biol 195(5):751–764.
7. Popp MW, Karssemeijer RA, Ploegh HL (2012) Chemoenzymatic site-specific labeling
of influenza glycoproteins as a tool to observe virus budding in real time. PLoS Pathog
8(3):e1002604.
Tafesse et al. www.pnas.org/cgi/content/short/1219909110
1 of 2
4
KBM7
SGMS1GT
% mol (of total lipids)
3
2
1
14
:0
1
16 5:
:0 0
-D
H
16
:0
18 16:
:0 1
-D
H
18
:0
20 18:
:0 1
-D
H
20
:0
20
:1
21
:0
21
:1
22
:0
22
:1
23
:0
23
:1
24
:0
24
:1
0
Sphingomyelin species
GT
Fig. S1. SM species of KBM7 and SGMS1 cells. Total lipids were extracted from KBM7 and SGMS1-null cells, and levels of SM species were analyzed by MS/MS
as described in Methods. Levels of SM species are given in mole percent of total lipids analyzed. Error bars, SD of two independent experiments.
A
0h
3h
Myr PI-PLC
% max
_
_
+
+
5h
Myr PI-PLC
_
+
_
+
_
_
+
+
Myr PI-PLC
_
+
_
+
_
+
_
+
Thy1.2-PE
Thy1.2-PE
% max
Thy1.2-PE
_
_
+
+
Class I-FITC
B
Class I-FITC
KBM7
37OC
0
0.5
1
SGMS1GT
37OC
O
4 C
3
3
Class I-FITC
0
0.5
1
3
4 OC
(h)
3
150
100
75
50
37
25
kDa
Fig. S2. Perturbation of sphingolipid biosynthesis modestly affects surface display of Thy1 but not overall protein secretion. (A) Myriocin-treated and control
EL4 cells were subjected to PI-PLC treatment for 1 h at room temperature, washed with PBS, and returned to 37 °C in fresh media. At the indicated time points,
cells were collected and costained with anti-Thy1-PE (Upper) and class I MHC-FITC (Lower) and analyzed by FACS. (B) Equal numbers of KBM7 and SGMS1GT cells
were pulse-labeled with [35S]-methionine/cysteine for 2 h and chased at 37 °C or kept on ice. Supernatants were collected and analyzed by SDS/PAGE and
autoradiography.
Tafesse et al. www.pnas.org/cgi/content/short/1219909110
2 of 2