The HIV lipidome: A raft with an unusual composition
Britta Brügger*, Bärbel Glass†, Per Haberkant*, Iris Leibrecht*, Felix T. Wieland*‡, and Hans-Georg Kräusslich†‡
*Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany; and †Department of Virology,
Universitätsklinikum Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany
Communicated by Kai Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, December 26, 2005 (received for review
December 10, 2005)
dihydrosphingomyelin 兩 nano-electrospray ionization tandem mass
spectrometry 兩 lipid analysis 兩 viral membrane
T
he nucleic acid and protein constituents of many viruses have
been identified, quantitated, and characterized in detail,
whereas a comprehensive and quantitative analysis of the lipid
composition, including molecular species of each lipid class, has
not been reported for any virus. Enveloped viruses acquire their
membrane by budding from a host cell membrane, but previous
reports already indicated that viral lipids may differ from those
of their respective budding membrane. It was suggested, therefore, that these viruses bud from membrane microdomains, and
this hypothesis has gained momentum in recent years in the
context of the lipid raft model (for review of lipid rafts, see ref.
1). Many viruses are now believed to bud from lipid rafts, with
this assignment being based on the coclustering of viral structural proteins with putative raft markers, their partitioning into
buoyant fractions after membrane extraction with cold detergent, and the sensitivity of virus release and兾or infectivity to
cholesterol extraction (2). The main method to operationally
define raft lipids and proteins was extraction with cold Triton
X-100. This methodology leads to artificial aggregation of raft
components, however, and therefore, can not be used to define
raft microdomains in natural cell membranes. The present
concept of lipid rafts is that they are dynamic assemblies of
sphingolipids, cholesterol, and raft proteins that associate and
dissociate on a rapid time scale. These assemblies can be induced
to coalesce from specific raft clusters usually by protein–protein
interactions, and these assemblies are the platforms that are used
in membrane trafficking, signaling, and virus budding (3, 4).
Most of the evidence for the existence of lipid rafts relies on
indirect methods, however, both in the analysis of cell and viral
membranes (5). The in vivo existence of lipid rafts remains
extremely controversial, because evidence for their presence in
intact biological membranes is still not conclusive, despite that
such evidence has been sought for more than a decade (6).
HIV type 1 (HIV-1) is an enveloped retrovirus, which buds
primarily from the plasma membrane of infected T cells.
HIV-1 morphogenesis is driven by the viral Gag polyprotein,
which has been shown to partially localize to detergentresistant membranes (DRMs) in infected cells. This result and
the observation that HIV-1 particles contain putative raft
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0511136103
proteins led to the suggestion that HIV-1 budding occurs from
lipid rafts (ref. 2 and references therein). Earlier support for
this hypothesis came from a comparative analysis of lipid
groups from HIV-1 particles and plasma membranes of producer cells that revealed an enrichment of cholesterol and
sphingomyelin (SM) in the virus (7, 8). Furthermore, cholesterol depletion impaired HIV-1 release and infectivity (9). A
cholesterol requirement for virus morphogenesis has also been
reported for alphaviruses (10), which are suggested not to bud
from lipid rafts (11), and the association of HIV-1 budding and
lipid rafts remains controversial.
Results and Discussion
Quantitative Lipid Analysis of HIV-1. To investigate HIV-1 envel-
opment, we performed a quantitative composition analysis of
viral lipids, including analysis of molecular species. HIV-1
particles were purified from the medium of the infected T cell
line, MT-4, by velocity gradient centrifugation. This protocol
yields essentially vesicle-free virus preparations (12). Lipid extracts from HIV-1 and from uninfected or infected MT-4 cells
were subjected to nano-electrospray ionization tandem mass
spectrometry (13). As control for purity, tissue culture supernatant from uninfected cells was subjected to the same procedure, and no lipid background signal was detectable in this case
(data not shown). No significant differences in lipid composition
were observed between infected and uninfected cells.
Table 1 shows that the phospholipid composition of HIV-1
differed significantly from that of MT-4 cells. Because of technical limitations in the homogenous preparation of plasma
membranes, the lipid composition was analyzed for total cellular
membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as major phospholipids of mammalian membranes,
were reduced in viral membranes by a factor of 2.7 and 2.1,
respectively. In contrast, SM and dihydrosphingomyelin
(DHSM) (collectively referred to as SMs), 1-alkenyl,2acylglycerophosphoethanolamine [referred to as plasmalogen
PE (pl-PE, plasmenylethanolamine)], and phosphatidylserine
(PS) were enriched in viral membranes by a factor of 3.2 (SMs),
2.1 (PS), and 1.7 (pl-PE) (for structures of lipids analyzed, see
Fig. 6, which is published as supporting information on the PNAS
web site). Cholesterol was also increased strongly in viral membranes: The ratio of cholesterol to total phospholipids was 0.39
in host and 0.83 in viral membranes (Fig. 1a). Likewise, monohexosylceramide (HC) was increased by a factor of 2.6 from
0.0015 mol % (host membranes) to 0.0038 mol % (virus).
Employing separation by thin layer chromatography of glycolipids extracted from MT-4 cells, the HC species was identified
as glucosylceramide (data not shown). Ceramide (Cer) was
reduced by a factor of 3.8 in viral membranes (0.004 mol % in
Conflict of interest statement: The communicating member K.S. is a founder in a small
start-up company called Jado Technologies that has raft technology as its specialty.
Abbreviations: Cer, ceramide; DHSM, dihydrosphingomyelin; DRM, detergent-resistant
membrane; FB1, fumonisin B1; HC, monohexosylceramide; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PS, phosphatidylserine; SM, sphingomyelin.
‡To whom correspondence may be addressed. E-mail: felix.wieland@urz.uni-heidelberg.de
or hans-georg.kraeusslich@med.uni-heidelberg.de.
© 2006 by The National Academy of Sciences of the USA
PNAS 兩 February 21, 2006 兩 vol. 103 兩 no. 8 兩 2641–2646
CELL BIOLOGY
The lipids of enveloped viruses play critical roles in viral morphogenesis and infectivity. They are derived from the host membranes
from which virus budding occurs, but the precise lipid composition
has not been determined for any virus. Employing mass spectrometry, this study provides a quantitative analysis of the lipid constituents of HIV and a comprehensive comparison with its host
membranes. Both a substantial enrichment of the unusual sphingolipid dihydrosphingomyelin and a loss of viral infectivity upon
inhibition of sphingolipid biosynthesis in host cells are reported,
establishing a critical role for this lipid class in the HIV replication
cycle. Intriguingly, the overall lipid composition of native HIV
membranes resembles detergent-resistant membrane microdomains and is strikingly different from that of host cell membranes.
With this composition, the HIV lipidome provides strong evidence
for the existence of lipid rafts in living cells.
Table 1. Phospholipid composition of MT-4 cells and HIV-1
PC
SM ⫹ DHSM
PE
pI-PE
PS
MT-4 cells
(mol % ⫾ SD)
HIV-1
(mol % ⫾ SD)
43.0 ⫾ 2.9
10.4 ⫾ 1.6
17.0 ⫾ 1.5
15.9 ⫾ 0.5
7.4 ⫾ 0.8
16.0 ⫾ 1.0
33.1 ⫾ 1.2
8.2 ⫾ 1.3
27.0 ⫾ 3.3
15.5 ⫾ 2.2
Lipids were extracted and analyzed for phospholipid content as described
in Materials and Methods. Values are expressed either as mol percentage of
a given phospholipid to total phosphate (MT-4 cells) or as mol percentage of
a given phospholipid to the total of all phospholipids quantified (HIV-1).
host and 0.001 mol % in viral membranes) (Fig. 1b). Lysobisphosphatidic acid (LBPA), a cone-shaped phospholipid,
which has been shown to be required for vesicle formation at the
late endosome (14), could not be quantified by mass spectrometry because it cannot be unequivocally distinguished from its
mass isomer phosphatidylglycerol.
An enrichment of sphingolipids and cholesterol is characteristic for DRM. A second hallmark of DRM is the enrichment of
saturated lipid species such as dipalmitoyl-PC (15). Quantitative
analysis of lipid subclasses revealed a 3.6-fold increase of saturated PC species in HIV-1 compared with the cellular membrane, resulting in a total of 40% saturated PC species in the
HIV-1 membrane, with dipalmitoyl-PC alone representing
⬇20% of total PC (see Fig. 7, which is published as supporting
information on the PNAS web site). This enrichment was
balanced by a reduction of diunsaturated and polyunsaturated
species (2.5- and 2.3-fold, respectively), whereas monounsaturated species remained constant with a contribution of 36–38%
(see Fig. 7b). The increase in saturated PC species is mainly
attributed to short chain (up to 32 C atoms in acyl chains) PC
species (see Fig. 7c), which were increased from 18% in cell
membranes to 41% in viral membranes, at the expense of both
medium (up to 36 C atoms) and long (ⱖ38 C atoms) chain PC
species. This tendency was also observed for PE, pl-PE, and PS,
albeit less pronounced (see Figs. 8–10, which are published as
supporting information on the PNAS web site). In contrast, no
significant changes with regard to saturation or fatty acid chain
length were observed for SM species (see Fig. 11, which is
published as supporting information on the PNAS web site).
DHSM Is Highly Enriched in HIV-1 Membranes. To determine the
molecular species distribution of PC and SMs, we performed
precursor ion scanning selecting for fragment ions of m兾z 184 Da
Fig. 2. Enrichment of DHSM 16:0兾PC in HIV-1. (a) Lipids of cells (Upper) and
virus (Lower) were subjected to mass spectrometer analysis. Precursor ion
scanning was performed by selecting for fragment ions of m兾z 184 Da. Major
peaks are labeled giving lipid class, number of total C atoms, and double bonds
in acylated fatty acids. Spectra are normalized to the highest peak in the
displayed mass range. (b) Quantitative analysis of SM and DHSM was performed as described in Materials and Methods. Data are displayed as molar
ratios of the indicated lipid species to PC. Ratios determined for host cell
membranes were set to 1. Error bars represent standard deviation of the
mean.
(⫹PREC 184). These ions are the most abundant fragment ions
of PC and SM and correspond to the positively charged choline
phosphate head group. Unexpectedly, we found a strong enrichment of the unusual sphingolipid DHSM in HIV-1 (Fig. 2). In
contrast to SM, DHSM does not contain a 4,5-trans double bond
in its sphinganine backbone. To validate the presence of DHSM,
we subjected the ions of interest to collision-induced fragmentation and to mild basic treatment, thereby confirming their
identity (for details see Fig. 12, which is published as supporting
information on the PNAS web site). The major species of SM and
Fig. 1. Lipid composition of MT-4 cells and HIV-1. Quantitative lipid analysis was performed as described in Materials and Methods. Data are displayed as molar
ratio of individual lipid classes to PC. Error bars represent standard deviation of the mean.
2642 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0511136103
Brügger et al.
Inhibition of Sphingolipid Biosynthesis Reduces HIV-1 Infectivity.
Previous studies have reported that inhibition of cholesterol
biosynthesis in HIV-1-producing cells or cholesterol extraction
from cell or virus membranes caused a decrease in virus production and infectivity (9, 22). To test whether sphingolipids are
also crucial for HIV-1 formation and兾or infectivity, we subjected
infected MT-4 cells to fumonisin B1 (FB1) treatment. FB1
blocks both the de novo synthesis of sphingolipids (by inhibiting
the synthesis of dihydroceramide, which serves as precursor to all
sphingolipid species) as well as the salvage pathway (23). HIV-1
purified from FB1-treated cells exhibited a significantly reduced
ratio of sphingolipids compared with PC, consistent with the
expected effect of the inhibitor. Reductions of 19% (Cer), 30%
(SMs), and 54% (HC), respectively, were observed for the
different sphingolipids (Fig. 3a).
This reduction of sphingolipids (which was also observed in
cellular membranes; data not shown) had no significant effect on
virus release (Fig. 3b). A strong effect on HIV-1 infectivity was
observed, however. HIV-1 released from FB1-treated cells was
reproducibly 4-fold less infectious than virus from untreated cells
(Fig. 3b). Accordingly, the concentration of sphingolipids in the
HIV-1 membrane appears to be important for maintaining viral
infectivity, although not being required for virus budding. Our
results indicate that changes in the lipid composition of biologBrügger et al.
Fig. 3. FB1 treatment of virus-producing cells reduces HIV-1 infectivity. HIV-1
infected MT-4 cells were cultured in the absence or presence of 50 M FB1. (a)
Quantitation of lipids from purified virus was performed by nano-electrospray
ionization tandem mass spectrometry. Values are expressed as molar ratios of
individual lipid classes to PC. (b) Quantitation of HIV-1 release (measured by
antigen ELISA; Left) and infectivity of cell-free virus (measured by infection of
TZM cells; untreated samples were set to 100%; Right). Bars 1 and 2 correspond
to two independent infections, each performed in triplicate. Error bars represent standard deviation of the mean.
ical membranes may only be tolerable within a narrow range,
because partial reduction of viral sphingolipids yielded a substantial decrease in HIV-1 infectivity. The conclusion that subtle
changes in lipid composition can lead to significant functional
alterations had been drawn previously from studies on transport
processes involving vesicular structures (24, 25). Future experiments will target specific sphinganine- and sphingosine-derived
lipids to correlate loss of HIV-1 infectivity with specific lipids
and to determine their mechanism of action. Our results with
FB1 suggest that lipid active drugs effectuating only subtle
changes in membrane composition have the potential to become
effective therapeutic agents against HIV.
Lipids of Native HIV-1 Membranes and DRM. Our analysis of HIV-1
lipid composition revealed a similar picture as previously reported for DRM, and we therefore performed a direct comparison of HIV-1 and DRM from infected MT-4 cells. DRM were
prepared by using the detergents Triton X-100 and Brij 96, which
differ in their ability to solubilize specific proteins and lipids,
with Triton X-100 being more stringent (26). Fractions after
isopycnic buoyant density centrifugation were analyzed regarding distribution of the DRM marker flotillin and of transferrin
receptor, which is not associated with DRM (Fig. 4a). Flotillin
was restricted to fraction 2 in the case of Triton X-100 extraction,
whereas transferrin receptor was found at the bottom of the
gradient (fractions 7–9), thus defining fraction 2 as DRM
fraction. Brij 96 was much less stringent, resulting in a broader
distribution of both flotillin and transferrin receptor (Fig. 4a
Lower). Fig. 4b shows that the ratio of SMs兾PC was strongly
increased in DRM from Triton X-100 as well as from Brij
96-treated cells (compared with total cell lipids) and was similar
PNAS 兩 February 21, 2006 兩 vol. 103 兩 no. 8 兩 2643
CELL BIOLOGY
DHSM in the viral and cellular membrane were SM d18:1兾16:0
and DHSM d18:0兾16:0, both containing an amide-linked palmitoyl group (Fig. 2a).
Quantitation of SM and DHSM species was performed by
employing the sensitive ⫹PREC 184 scan (Fig. 2). There was no
difference in the relative abundance of choline phosphate fragment ions generated from lipids containing either a sphingosine
or a sphinganine backbone (Fig. 12d and ref. 16), and nonnatural
SM species could therefore be used for quantitation of both
lipids. As shown in Fig. 2b, the major species of both SM and
DHSM were highly enriched in viral compared with cellular
membranes: The molar ratio of SM 16:0兾PC was increased
almost 8-fold in HIV-1 (0.12 versus 0.95), and the molar ratio of
DHSM 16:0兾PC was increased by a factor of almost 15 (0.028
versus 0.41). Thus, the dihydro species of SM is enriched in
HIV-1 even higher than SM, and the molecular species Npalmitoyl-DHSM alone constitutes ⬇10% mol of total HIV-1
phospholipids. We also confirmed the presence of dihydroceramide as a potential precursor of DHSM as well as dihydromonohexosylceramide, but quantitation was not possible because of
the low yield of the relevant fragment ions (16).
DHSM has not been reported as a component of HIV-1, which
is not surprising given that data for other sphingolipid species in
the HIV-1 membrane are also not available. So far, DHSM was
described as a major constituent of biological membranes only in
human lens extracts, where it accounts for 50% of all phospholipids (17). The function of DHSM is currently not known.
DHSM in the cholesterol-rich membranes of the eye lens has
been suggested to contribute to a greater resistance to oxidation
and to the formation of cholesterol crystallites (18). Biophysical
studies on liposomes comparing acyl-chain-matched SM and
DHSM have shown that DHSM leads to formation of more
ordered membranes with a higher melting temperature because
of its less polar nature (18–20). Based on these results, DHSM
was suggested to function as membrane organizer in laterally
condensed liquid-ordered membrane domains, such as lipid
rafts. Accordingly, the high amount of DHSM in the SM- and
cholesterol-rich HIV-1 membrane may contribute to membrane
order and physical stability of the extracellular virion but may
also be important for resistance to oxidation. Conceivably,
pl-PE, which is also enriched in the HIV-1 membrane, may
contribute to this resistance as well, as plasmalogens have been
discussed to function as antioxidants (for review, see ref. 21).
Fig. 4. DRM from infected cells and native HIV-1 membranes show similar enrichment of SM and DHSM. HIV-1 particles and DRM were isolated as described
in Materials and Methods. The SM, DHSM, and PC content in virus membranes and DRM was determined by nano-electrospray ionization tandem mass
spectrometry, whereas the distribution of selected proteins was determined by Western blot. (a) DRM obtained by cold extraction of HIV-1 infected MT-4 cells
with Triton X-100 (TX100, Upper) or Brij 96 (Lower) were analyzed for raft (flotillin 1) and nonraft (transferrin receptor, TfR) marker distribution. (b) SMs to PC
ratios in total cell membranes, HIV-1, and DRM obtained with TX100 or Brij 96. (c) SM (16:0) and DHSM (16:0) to PC ratios in HIV-1 and DRM. (d) Extracts from
infected MT-4 cells (lane 1), DRM from infected cells (obtained by cold TX100 extraction; lanes 2– 4), and purified HIV-1 (lanes 5–7) were subjected to Western
blot analysis detecting flotillin or gp41. The amount of cell extracts loaded was normalized according to cell number (105 cells in lane 1; DRM from 3 ⫻ 105, 2 ⫻
105, and 105 cells in lanes 2, 3 and 4, respectively), and the amount of virus loaded was normalized according to total PC and SM content of DRM and HIV-1
preparations with equal amounts of lipids loaded in lanes 4 and 6 (lane 5, 500% of lane 6; lane 7, 50% of lane 6). Error bars represent standard deviation of the
mean.
to native HIV-1 lipids. This result was also observed when the
ratios of SM 16:0兾PC and DHSM 16:0兾PC were analyzed (Fig.
4c), indicating that DHSM was enriched to a similar extent in
native HIV-1 membranes and DRM as well. Thus, the HIV-1
membrane shows lipid ratios very similar to DRM, despite being
prepared without any detergent treatment.
To compare the segregation of lipids and proteins into DRM
and viral membranes, respectively, we analyzed flotillin and the
HIV-1 transmembrane glycoprotein (gp)41 (and its precursor
gp160) in the various fractions (Fig. 4d). In the case of flotillin,
⬇50% was recovered in the DRM fraction from Triton X-100
extracted cells (Fig. 4d Lower, compare lanes 1 and 3). Recovery
appeared to be lower for HIV-1 gp41 (Fig. 4d Upper, compare
lanes 1 and 3), whereas the uncleaved precursor gp160 was
virtually absent from DRM. A very different result was observed
for extracts from native HIV-1, where gp41 was strongly enriched (Fig. 4d, compare lanes 4 and 6), and flotillin was not
detectable. This finding differs from previous studies reporting
the incorporation of other DRM-associated proteins into HIV-1
(for review, see ref. 2). The segregation of flotillin from HIV
membranes is consistent with the viral budding platform being
formed by coalescence of a subset of preexisting lipid microdomains, which may be induced by the viral structural proteins.
Because the native HIV-1 membrane resembles a DRM, we
analyzed lipid and protein distribution after isopycnic density
gradient centrifugation of Triton X-100 extracts from purified
HIV-1 particles. As observed for cell extracts, HIV-1 lipids and
proteins segregated into buoyant and nonbuoyant fractions. The
distribution into detergent-sensitive and detergent-resistant
fractions was similar for SMs and gp41, with approximately
one-third in the detergent-resistant fraction. This result indicates
that there is no obvious preference of the viral fusion machinery
2644 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0511136103
for membrane domains of different detergent sensitivity. The
SM 16:0兾PC and DHSM 16:0兾PC ratios were similar for native
HIV-1 membranes, DRM from HIV-1, and DRM from infected
cells.
The HIV-1 Lipidome: Its Relation to Lipid Rafts. Our quantitative
analysis of the lipids of highly purified native HIV-1 allows us to
calculate an estimate for the numbers of individual lipid molecules per virus particle, even with respect to their molecular
species distribution (the HIV-1 lipidome). We estimate that this
analysis includes ⬎95% of all HIV-1 lipids based on prior
determination of other membranes (7, 8), and future experiments will be directed at quantifying minor lipid groups (e.g.,
phosphatidylinositol phosphates). A graphical representation of
the lipid composition of HIV-1 membranes compared with the
surrounding membrane of the producer cell is shown in Fig. 5.
Quantitation of HIV-1 lipids and structural proteins from the
same sample provides a quantitative description of lipid molecules per virion. Assuming 4,900 Gag proteins (27), this calculation yielded ⬇296,000 lipid molecules per average HIV-1
particle for two independent preparations, with the distribution
of lipids shown in Table 2 (for details of calculation, see
Supporting Text, which is published as supporting information on
the PNAS web site). The number of lipids per virion can also be
calculated from the viral diameter and the average footprint of
a lipid molecule. With an average outer HIV-1 diameter of 145
nm (28), this calculation yields a theoretical number of 255,000
lipids per particle assuming a 0.5 nm2 surface area per lipid
molecule, which is in excellent agreement with the experimentally determined number.
The observation that the lipid composition of HIV-1 is very
similar to that proposed for lipid microdomains (rafts) (29) in at
Brügger et al.
The lipid composition of HIV particles. For details, see text.
least five different aspects (enriched in saturated lipids, PS,
pl-PE, cholesterol, and sphingolipids) strongly supports the
hypothesis that HIV-1 buds from cellular membrane microdomains. The absence of the bona fide raft marker flotillin from
pure HIV-1 preparations is in line with the concept that HIV
budding is a specific raft clustering process, whereas DRM
represents general aggregations of raft microdomains. HIV
proteins are probably interacting with rafts, and interactions
between them may drive the clustering process. It will be a future
challenge to identify the viral gene products that determine
HIV-1 lipid composition and to define potential host cell
differences and their influence on infectivity. Most importantly,
however, the lipid composition of highly purified native HIV-1,
prepared in the absence of any detergent, is among the strongest
evidence for the existence of raft-like lipid microdomains in
biological membranes of living cells.
Materials and Methods
Materials. FB1 and Brij 96 were obtained from Sigma. Sphingosylphosphorylcholine was obtained from Matreya (Pleasant
Gap, PA). Unsaturated PC species for transphosphatidylation
and SM (porcine brain and chicken egg) were purchased from
Avanti Polar Lipids, and fatty acids and Triton X-100 were from
Merck.
Table 2. The lipid composition of HIV-1
Lipid molecules per average HI virion
PC
SM
DHSM
PE
pl-PE
PS
Chol
Cer
HC
For details, see Supporting Text.
Brügger et al.
26,000
37,000
17,000
13,000
44,000
25,000
134,000
160
600
Antibodies. Monoclonal mouse anti-human transferrin receptor
antibody (clone H68.4) was from Zymed. The polyclonal rabbit
anti-flotillin-1 antibody was kindly donated by J. B. Helms
(University of Utrecht, The Netherlands). Monoclonal antibody
against HIV-1 gp41 was derived from cell culture media of
Chessie 8 cells obtained from George Lewis (30) through the
National Institutes of Health AIDS research and reagent reference program. Polyclonal rabbit serum against the HIV-1 capsid
protein had been raised against purified protein.
Cell Culture and Virus Purification. MT-4 cells (31) were maintained
at 37°C and 5% CO2 in RPMI 1640 medium supplemented with
10% heat-inactivated FCS, antibiotics, 4 mM L-glutamine, and 5
mM Hepes. Cells were infected with HIV-1 strain NL4–3 (32),
and the virus was harvested from cocultures of infected and
uninfected cells before cytopathic effects were observed as
described in ref. 12. For inhibitor treatment, FB1 in DMSO was
added at a final concentration of 50 M.
HIV-1 purification was performed as described (12). Briefly,
particles were concentrated from cleared media by centrifugation through a cushion of 20% (wt兾wt) sucrose in PBS. Concentrated HIV-1 was further purified by velocity gradient centrifugation on an Optiprep gradient (Axis-Shield, Oslo,
Norway). The visible virus band was collected and pelleted
yielding a 1,800-fold concentration compared with the initial
volume. Virus titers were determined on TZM cells containing
a -galactosidase gene under control of the HIV-1 promoter as
described (33).
Quantitative Analysis of HIV-1 Release. Virus release was quanti-
tated by ELISA of cleared culture supernatants from infected
MT-4 cells detecting the HIV-1 capsid protein p24. Quantitative
Western blot analysis was performed by using the Odyssey
infrared imaging system from Li-Cor (Lincoln, NE).
Isolation of DRM. Infected or uninfected MT-4 cells (1 ⫻ 107) were
washed and extracted on ice for 15 min in 0.3 ml of lysis buffer
(50 mM Tris䡠HCl, pH 7.4兾150 mM NaCl兾5 mM EDTA兾1 mM
DTT兾1% Triton X-100). The lysate was dounced ten times,
mixed with 0.6 ml of Optiprep (Axis-Shield) and overlaid with
PNAS 兩 February 21, 2006 兩 vol. 103 兩 no. 8 兩 2645
CELL BIOLOGY
Fig. 5.
2.5 ml 28% Optiprep in lysis buffer, followed by 0.6 ml of lysis
buffer in a SW60 tube. Tubes were centrifuged at 126,000 ⫻ g for
3 h at 4°C. Eight fractions (450 l each) were collected from the
top. Western blots of gradient fractions were probed with
antibodies against flotillin-1, transferrin receptor, or HIV-1
gp41. DRM fractions were directly subjected to mass spectrometer analysis. DRM preparation from HIV-1 particles was as
described above, except that purified virus from 600 ml of culture
supernatant from infected MT-4 cells was used as input material.
Lipid Analysis. Lipid extractions were performed according to
and N-palmitoyl-Cer, respectively. Quantitation of PE and PS
was performed by neutral loss scanning, selecting for a neutral
loss of 141 Da or 185 Da (positive ion mode), respectively, with
a collision energy of 20 eV. pl-PE quantitation was performed
by precursor ion scanning for fragment ion m兾z 196 Da
(negative ion mode; collision energy of 40 eV). Unsaturated
PE and PS standards were synthesized and purified via HPLC
as described (36). Quantitative analyses were performed as
described (13, 37). Phosphate determination was performed
according to Rouser (38).
the method of Bligh and Dyer (34). Quantitation of PC, SM,
DHSM, and cholesterol was performed as described (35). HC
and Cer scanning was performed by precursor ion scanning for
fragment ion 264 Da (positive ion mode) at a collision energy
of 35 eV (1 eV ⫽ 1.602 ⫻ 10⫺19 J) (HC) or 30 eV (Cer).
Quantitative data given refer to major species N-palmitoyl-HC
We thank Walter Nickel [Heidelberg University Biochemistry Center
(BZH)] for helpful comments and critical reading of the manuscript. This
work was supported by Deutsche Forschungsgemeinschaft Grants
SPP1175 (to B.B. and F.T.W.) and SFB638, A9 (to H.-G.K.).
1. Simons, K. & Vaz, W. L. (2004) Annu. Rev. Biophys. Biomol. Struct. 33,
269–295.
2. Ono, A. & Freed, E. O. (2005) Adv. Virus Res. 64, 311–358.
3. Kusumi, A., Koyama-Honda, I. & Suzuki, K. (2004) Traffic 5, 213–230.
4. Simons, K. & Toomre, D. (2000) Nat. Rev. Mol. Cell Biol. 1, 31–39.
5. Munro, S. (2003) Cell 115, 377–388.
6. Lagerholm, B. C., Weinreb, G. E., Jacobson, K. & Thompson, N. L. (2005)
Annu. Rev. Phys. Chem. 56, 309–336.
7. Aloia, R. C., Jensen, F. C., Curtain, C. C., Mobley, P. W. & Gordon, L. M.
(1988) Proc. Natl. Acad. Sci. USA 85, 900–904.
8. Aloia, R. C., Tian, H. & Jensen, F. C. (1993) Proc. Natl. Acad. Sci. USA 90,
5181–5185.
9. Ono, A. & Freed, E. O. (2001) Proc. Natl. Acad. Sci. USA 98, 13925–13930.
10. Lu, Y. E. & Kielian, M. (2000) J. Virol. 74, 7708–7719.
11. Scheiffele, P., Rietveld, A., Wilk, T. & Simons, K. (1999) J. Biol. Chem. 274,
2038–2044.
12. Welker, R., Hohenberg, H., Tessmer, U., Huckhagel, C. & Kräusslich, H. G.
(2000) J. Virol. 74, 1168–1177.
13. Brügger, B., Erben, G., Sandhoff, R., Wieland, F. T. & Lehmann, W. D. (1997)
Proc. Natl. Acad. Sci. USA 94, 2339–2344.
14. Matsuo, H., Chevallier, J., Mayran, N., Le Blanc, I., Ferguson, C., Faure, J.,
Blanc, N. S., Matile, S., Dubochet, J., Sadoul, R., Parton, R. G., Vilbois, F. &
Gruenberg, J. (2004) Science 303, 531–534.
15. Fridriksson, E. K., Shipkova, P. A., Sheets, E. D., Holowka, D., Baird, B. &
McLafferty, F. W. (1999) Biochemistry 38, 8056–8063.
16. Sullards, M. C. (2000) Methods Enzymol. 312, 32–45.
17. Byrdwell, W. C., Borchman, D., Porter, R. A., Taylor, K. G. & Yappert, M. C.
(1994) Invest. Ophthalmol. Vis. Sci. 35, 4333–4343.
18. Epand, R. M. (2003) Biophys. J. 84, 3102–3110.
19. Kuikka, M., Ramstedt, B., Ohvo-Rekila, H., Tuuf, J. & Slotte, J. P. (2001)
Biophys. J. 80, 2327–2337.
20. Nyholm, T., Nylund, M., Soderholm, A. & Slotte, J. P. (2003) Biophys. J. 84,
987–997.
21. Nagan, N. & Zoeller, R. A. (2001) Prog. Lipid Res. 40, 199–229.
22. Campbell, S. M., Crowe, S. M. & Mak, J. (2002) AIDS 16, 2253–2261.
23. Wang, E., Norred, W. P., Bacon, C. W., Riley, R. T. & Merrill, A. H., Jr. (1991)
J. Biol. Chem. 266, 14486–14490.
24. Stüven, E., Porat, A., Shimron, F., Fass, E., Kaloyanova, D., Brügger, B.,
Wieland, F. T., Elazar, Z. & Helms, J. B. (2003) J. Biol. Chem. 278, 53112–
53122.
25. Wang, Y., Thiele, C. & Huttner, W. B. (2000) Traffic 1, 952–962.
26. Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A. & Simons, K. (2003) Proc.
Natl. Acad. Sci. USA 100, 5795–5800.
27. Briggs, J. A., Simon, M. N., Gross, I., Kräusslich, H. G., Fuller, S. D., Vogt,
V. M. & Johnson, M. C. (2004) Nat. Struct. Mol. Biol. 11, 672–675.
28. Briggs, J. A., Wilk, T., Welker, R., Kräusslich, H. G. & Fuller, S. D. (2003)
EMBO J. 22, 1707–1715.
29. Pike, L. J., Han, X., Chung, K. N. & Gross, R. W. (2002) Biochemistry 41,
2075–2088.
30. Abacioglu, Y. H., Fouts, T. R., Laman, J. D., Claassen, E., Pincus, S. H., Moore,
J. P., Roby, C. A., Kamin-Lewis, R. & Lewis, G. K. (1994) AIDS Res. Hum.
Retroviruses 10, 371–381.
31. Harada, S., Koyanagi, Y. & Yamamoto, N. (1985) Science 229, 563–566.
32. Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A. &
Martin, M. A. (1986) J. Virol. 59, 284–291.
33. Müller, B., Daecke, J., Fackler, O. T., Dittmar, M. T., Zentgraf, H. &
Kräusslich, H. G. (2004) J. Virol. 78, 10803–10813.
34. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911–917.
35. Brügger, B., Graham, C., Leibrecht, I., Mombelli, E., Jen, A., Wieland, F. &
Morris, R. (2004) J. Biol. Chem. 279, 7530–7536.
36. Koivusalo, M., Haimi, P., Heikinheimo, L., Kostiainen, R. & Somerharju, P.
(2001) J. Lipid Res. 42, 663–672.
37. Brügger, B., Sandhoff, R., Wegehingel, S., Gorgas, K., Malsam, J., Helms, J. B.,
Lehmann, W. D., Nickel, W. & Wieland, F. T. (2000) J. Cell Biol. 151, 507–518.
38. Rouser, G., Fleischer, S. & Yamamoto, A. (1970) Lipids 5, 494–496.
2646 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0511136103
Characterization of DHSM. See Supporting Text.
Brügger et al.