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Biochemical Society Transactions (2013) Volume 41, part 1
Palmitoylation of influenza virus proteins
Michael Veit*1 , Marina V. Serebryakova† and Larisa V. Kordyukova†
*Department of Immunology and Molecular Biology Veterinary Faculty, Free University, Philippstr. 13, 10115 Berlin, Germany, and †Belozersky Institute of
Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1, Bldg 40, 119991 Moscow, Russia
Abstract
Influenza viruses contain two palmitoylated (S-acylated) proteins: the major spike protein HA
(haemagglutinin) and the proton-channel M2. The present review describes the fundamental biochemistry
of palmitoylation of HA: the location of palmitoylation sites and the fatty acid species bound to HA. Finally,
the functional consequences of palmitoylation of HA and M2 are discussed regarding association with
membrane rafts, entry of viruses into target cells by HA-mediated membrane fusion as well as the release
of newly assembled virus particles from infected cells.
Palmitoylated proteins of influenza virus
Influenza viruses are enveloped viruses found in the
Orthomyxoviridae family. Their membrane is lined from
beneath by the matrix protein M1, which in turn envelopes
the viral genome. In influenza A and B viruses there are two
viral spikes embedded in the envelope: HA (haemagglutinin),
which catalyses virus entry by binding to sialic acid moieties
present on the host cell surface and by performing fusion of
viral with endosomal membranes and NA (neuraminidase),
which is required for the release of virus particles by removing
potential receptors from infected cells. In the influenza C
virus, all three activities (receptor-binding and -destroying,
and membrane fusion) are combined in one spike, which
is designated HEF (HA-esterase fusion glycoprotein). Virus
particles also contain minor amounts of a proton channel,
which is called M2 in influenza A virus and BM2 and CM2
in influenza B and C virus.
HA and HEF, as well as M2 and CM2, are palmitoylated at
cytoplasmic and transmembrane cysteine residues, whereas
the other viral proteins lack any lipid modifications. This
review describes the biochemistry of HA acylation and how
the modification affects targeting of HA and M2 to rafts
and (probably as a consequence) virus budding and virus
entry. Several recent reviews cover related topics, such as
palmitoylation of other viral proteins [1], raft association
of influenza virus proteins [2], budding of influenza virus
particles [3] and HA-catalysed membrane fusion [4].
Fatty acid species bound to HA and HEF
HA (as seen in Figure 1) and HEF are trimeric type I
transmembrane glycoproteins with an N-terminal signal
Key words: budding, haemagglutinin (HA), influenza virus, M2, membrane fusion, virus
taxonomy.
Abbreviations used: CRAC, cholesterol recognition/interaction amino acid consensus;
DRM, detergent-resistant membrane; FRET, fluorescence resonance energy transfer; HA,
haemagglutinin; HEF, HA-esterase fusion glycoprotein; NA, neuraminidase; NCBI, National Center
for Biotechnology Information; NP, nucleoprotein; TMR, transmembrane region; VLP, virus-like
particle.
1
To whom correspondence should be addressed (email [email protected]).
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Authors Journal compilation peptide, a large ectodomain, a single TMR (transmembrane
region) and a short cytoplasmic tail. HA from influenza A is
highly variable; 17 antigenic subtypes have been isolated so
far, whereas HA from influenza B virus and especially HEF
are less variable. HA from influenza A virus is acylated at
three cysteine residues, two are located in the cytoplasmic
domain and one at the end of the TMR, whereas a cysteine
present in some subtypes in the middle of the TMR is
not acylated [5–7]. H11 subtype HA contains an additional
cytoplasmic cysteine, which is also used as acylation site [8]
(Table 1). HA from influenza B virus is palmitoylated at
two cysteine residues in its cytoplasmic tail and HEF, which
contains a very short cytoplasmic tail, is palmitoylated at a
single cysteine at the end of the TMR [9,10].
It has been shown early on that [3 H]palmitic acid, which
was used to identify acylated proteins, can be converted into
other fatty acid species of different chain length, these are
then attached to the acylprotein. However, it was not known
for proteins having multiple acylation sites whether each
site contains the same proportion of fatty acids or whether
specific cysteine residues are acylated predominantly with a
particular carbon chain. Advancements in MS have recently
allowed for quantitative analysis of fatty acid species linked
to individual acylation sites (reviewed in [11]). The increase
in mass of an acylated protein compared with the unmodified
protein is rather small, therefore only fragments of viral
spikes can be accurately measured. Purified virus particles
are digested with proteases, which remove the ectodomain
from the spikes, and the hydrophobic membrane-anchoring
fragments are extracted with chloroform/methanol and
directly analysed by MS. Tandem-MS sequencing of peptides
can be used to both prove their amino acid sequence and
identify the acylated cysteine residues [12].
These studies revealed a striking difference between HA
of influenza B virus that contains 97 % palmitate and
HEF of influenza C virus that is predominantly (88 %)
stearoylated (Table 1), confirming previous data obtained
by less sophisticated methods [13]. HAs of the influenza
A virus contain a mixture of palmitate and stearate. MS
analysis of recombinant viruses with deletions of individual
Biochem. Soc. Trans. (2013) 41, 50–55; doi:10.1042/BST20120210
Regulation of Protein Trafficking and Function by Palmitoylation
Figure 1 Palmitoylated proteins of influenza A virus
HA of influenza A virus is S-acylated at two cytoplasmic cysteine
residues with palmitate (wavy black line) and at a transmembrane
cysteine residue primarily with stearate (wavy brown line). The fatty
acids (together with hydrophobic amino acids at the beginning of the
TMR) target HA to large membrane-rafts (grey), which are the budding
sites of the virus. M2 is palmitoylated at a cysteine residue present in
an amphiphilic helix in its cytoplasmic tail. S-acylation together with
cholesterol binding (black star) are thought to target M2 to the edge of
the budozone. The amphiphilic helix is then thought to insert into the
membrane to induce curvature. Note that HA is a trimer and M2 is a
tetramer, which forms a proton channel.
cysteine residues, as well as tandem-MS sequencing, revealed
the surprising result that stearate is exclusively attached to
the cysteine positioned in the TMR of HA [14].
Since this initial observation [15], 40 HAs from 14 subtypes
have been analysed by MS [8,16]. The percentage of stearate
in all HA variants differs from 35 % (meaning that each
TMR cysteine contains stearate) to 12 % (indicating that
only one out of three TMR cysteine residues is stearoylated).
Interestingly, HAs that are present in virus strains isolated
from humans contain less stearate compared with HAs
isolated from mammals or birds. One reason might be that
other viral proteins, especially M1, which is much less variable
than HA, but contains host-specific amino acid substitutions,
affect acylation of HA, but we could not detect variability in
the stearate content of HA if internal proteins were exchanged
between viruses [16a].
What might be the main molecular signal that determines
preferential attachment of stearate to a cysteine residue, its
sequence context or its location relative to the membrane
span? At present, either bioinformatic sequence comparisons
have not revealed obvious amino acid peculiarities between
all analysed HA sequences that might be responsible for the
variations in the amount of HA-bound stearate or they are
located outside of the considered C-terminal area. An analysis
of H9 subtype HA, which contains only cytoplasmic cysteine
residues and very little stearate (4 %), suggests the effect of
location (Table 1). However, individual amino acids in the
vicinity of the TMR cysteine also affect fatty acid selection,
since HAs containing the same spacing of cysteine residues
differ in the amount of attached stearate. For example, only
one amino acid difference, a conservative exchange in the
middle of the TMR (highlighted in Table 1), exists between
the anchoring segments of HA from an equine and human
H3, but 30 % compared with 18 % of stearate was attached
to HA of those strains. An even larger difference is observed
between human H1 HA (12 %) and avian H5 HA (29 %),
although their cytoplasmic tails and the four amino acids
upstream of the TMR cysteine are identical. In addition, two
H1 HAs from duck and human strains possessing the same
amino acid sequence within the whole anchoring segment
showed a considerable variation in their stearate content
(18 % compared with 12 %, Table 1). Alternatively, since
the amount of HA [relative to NP (nucleoprotein) or M1]
present in virus particles varies largely between virus strains,
it might be that overexpression of HA might saturate the
stearoylation machinery of the cell if HA is expressed in very
high amounts and then the cysteine residues in the TMR
become palmitoylated.
The specific attachment of stearate leads to speculation on
the enzymology of acylation of HA. Members of the DHHC
family, polytopic membrane proteins containing a
DHHC (Asp-His-His-Cys) motif within one of their
cytoplasmic domains, were shown to palmitoylate cellular
proteins [17], but a DHHC protein that acylates influenza
HA (or other viral proteins) has not been identified. Alternatively, S-acylation of proteins can occur via a non-enzymatic
mechanism. Some cellular proteins are palmitoylated, at least
in vitro, at authentic sites in the absence of any enzyme source
[18,19]. In addition, peptides composed of unnatural β- or
D-amino acids are palmitoylated on microinjection into cells
indicating that the hallmarks of classical enzyme reactions,
substrate recognition and specificity do not exist [20].
The site-specific attachment of stearate to HA argues
against a purely non-enzymatic reaction mechanism. Acylation without an enzyme would not show any preference for
a particular fatty acid, but should reflect the concentration
of individual acyl-CoAs present in the membrane where
acylation occurs. Since HEF and HA are both acylated at
a late endoplasmic reticulum or early Golgi compartment
[10,21], it is more likely that individual enzymes exist
that differ in their acyl-CoA specificities, as recently
demonstrated for DHHC 2 and 3 [22]. One can imagine
that the active site of a DHHC protein with a preference for
stearoyl-CoA might penetrate deeper into the membrane to
attach stearate to a transmembrane cysteine compared with
an enzyme with specificity for palmitoyl-CoA.
Further MS analysis revealed that other viral glycoproteins,
such as F of Newcastle disease virus and E1 of Semliki Forest
virus, are also acylated with stearate at a transmembrane
cysteine [23]. Thus site-specific attachment of stearate is a
common feature of viral spike proteins and, since viruses
hijack the cellular acylation machinery, it also probably
occurs on surface receptors of the cell.
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Biochemical Society Transactions (2013) Volume 41, part 1
Table 1 Amino acid sequences and amount of stearate of HA and HEF from various influenza viruses
Data determined by MALDI–TOF-MS (matrix-assisted laser-desorption ionization–time-of-flight MS) from [8,14]. TMRs are underlined, acylated
cysteine residues are highlighted in grey, the amino acid patterns discussed in the text are highlighted. a Remainder is palmitate; results are
represented as means±S.D.
Function of S-acylation of HA
HA is a highly variable molecule with very low amino
acid conservation (≈20 %) through all subtypes. However,
comparison of all HA sequences present in the NCBI
(National Center for Biotechnology Information) database
(≈17000) showed that each molecule contains three, some
even four, cysteine residues located in the cytoplasmic domain
and at the cytosol-facing end of the TMR. Thus, assuming
these cysteine residues (and the attached fatty acids) would
not play an essential role for the life cycle of influenza viruses,
it is hard to conceive why they have not been exchanged by
similar amino acids during evolution of influenza viruses.
Indeed, it was impossible to generate influenza virus mutants
with two or three HA palmitoylation sites removed, implying
that this modification is essential for virus growth [24–26].
S-acylation of HA as raft-targeting signal
Palmitoylation is one of the best characterized signals
for association of proteins with rafts, heterogeneous lipidassemblies enriched in cholesterol and sphingolipids [27].
Raft association of a protein is often deduced from the
partitioning of a protein into DRMs (detergent-resistant
membranes). Using such assays, it has been shown that
deletion of palmitoylation sites from HA reduces their
partition into DRMs [24,25].
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Authors Journal compilation We used FRET (fluorescence resonance energy transfer)
to show that HA, fused at its cytoplasmic tail to CFP (cyan
fluorescent protein), clusters with an established marker
for inner leaflet rafts, double-acylated yellow fluorescent
protein [28]. Furthermore, an artificial HA-derived FRET
probe, consisting of a signal peptide, a fluorescent protein
and the transmembrane as well as cytoplasmic domain of
HA [29], clusters with a glycolipid-anchored protein, an
established marker for rafts of the outer leaflet. For both HA
constructs clustering was significantly reduced when all three
palmitoylation sites were removed from HA. Removing the
palmitoylation sites from HA slightly increased its mobility
in the plasma membrane. However, HA diffuses much slower
in comparison with the double acylated raft-marker indicating that association of HA with rafts is a dynamic process [28].
The concept that association of HA with rafts concentrates
the protein in the plasma membrane might explain how
protein palmitoylation might influence both assembly
of virus particles as well as the membrane fusion activity of
HA. Expression of HA at the plasma membrane organizes
a large raft domain [30,31], that is thought to provide a
platform for virus assembly and budding [32], enriching the
viral components to facilitate their interactions. Likewise,
the membrane fusion activity of HA critically depends on
its surface density. Hence, clustering of HA in rafts might
yield a concentration high enough to support fusion.
Regulation of Protein Trafficking and Function by Palmitoylation
Interestingly, HEF of influenza C virus is completely
soluble in cold detergent [33]. Thus S-acylation is not
sufficient to cause raft-localization of a viral glycoprotein.
Even the attachment of several fatty acids does not necessarily
cause raft-localization of a spike protein. The E1/E2
heterotrimer of Semliki Forest virus contains 15 covalently
linked acyl chains, six of them being stearate, but the spike
protein is nevertheless not associated with DRMs [23].
Involvement of S-acylation of HA in virus
assembly
HA seems to be a key player for influenza virus assembly and
budding; it organizes the viral budozone, which is a large raft
domain [30,31]. If HA is expressed from a plasmid, VLPs
(virus-like particles) are released that are morphologically
very similar to authentic virions. Co-expression of M1 leads
to its inclusion into VLPs, but it is much less efficient if the
cytoplasmic tail or the acylation sites were deleted from HA
[34]. In the context of virus infection, viruses lacking this
part of HA were found to have severe defects in genome
packaging as well as irregular morphology [35]. Direct
evidence suggesting that palmitoylation of HA is involved
in virus release was obtained for H3 subtype HA [24].
Virus particles containing HA with deleted palmitoylation
sites revealed defects in replication and incorporated reduced
amounts of the internal components NP and M1. Removal of
the palmitoylated cytoplasmic cysteine residues had a greater
effect on budding compared with exchange of the stearoylated
cysteine in the TMR, but the effect was opposite when
association with DRMs was analysed. Importantly, exchange
of the M1 protein by that of a different influenza virus
restored assembly of viruses with non-palmitoylated HA.
Involvement of S-acylation in HA-catalysed
membrane fusion
Upon virus entry and exposure to low pH levels, HA
undergoes a conformational change that catalyses the fusion
of viral and endosomal membranes [4]. Membrane fusion
assumingly proceeds via a hemifusion stage, where only the
outer leaflets of the two fusing membranes are connected.
Then a fusion pore in the membrane opens, flickers and
ultimately dilates. The hemifusion stage is characterized
by mixing lipids between the two membranes, solutes of
the two compartments are not exchanged before fusion is
complete.
Lipid mixing was not impaired in cells expressing nonacylated HA from all influenza virus strains examined suggesting that palmitoylation is not required for hemifusion. In
accordance, glycolipid-anchored HA catalyses hemifusion,
but not full fusion. This indicates that the TMR and the
cytoplasmic tail are required for this process [36].
Inconsistent data have been published concerning the effect
of acylation site removal on the ability of HA to cause full
fusion. It was reported that non-acylated H2 and H3 subtype
HA mediate cell–cell fusion or show unperturbed transfer
of aqueous dyes into HA-expressing cells [6,24]. In contrast,
non-acylated HA mutants from the H1, H7, another HA
from H2 subtype and from influenza B virus show impaired
fusion pore or syncytium formation [5,9,25,37]. Usually,
removal of cytoplasmic palmitoylation sites, especially the
most C-terminal one, which is surrounded by conserved
hydrophobic residues, had a more severe effect on fusion
compared with stearoylation of the transmembrane cysteine.
S-acylation of M2 and its function in virus
budding
M2 (purple structure in Figure 1), is a tetrameric proton
channel activated by acidic pH levels, the action of which
is important for genome unpacking during virus entry. In
each monomer, the first 24 amino acids form the unglycosylated ectodomain, the following 19 residues are the
TMR, the remaining 54 residues build up the cytoplasmic
tail. The sequence immediately following the TMR shapes
a membrane-parallel amphiphilic helix. A cysteine residue
in the helix is post-translationally acylated, primarily with
palmitate and probably in the same compartment as HA
[38,39]. In addition, the helix contains CRAC (cholesterol
recognition/interaction amino acid consensus) motifs [40].
CM2 from influenza C virus is also palmitoylated within a
(predicted) amphiphilic region [41] that, however, does not
contain cholesterol-binding motifs. BM2 from the influenza
B virus is neither palmitoylated nor does bioinformatic
analysis (using http://heliquest.ipmc.cnrs.fr/) predict an
amphiphilic helix in the cytoplasmic tail.
Previously, M2 has been implicated in the ultimate step
in virus budding: the scission of the virus particle from the
plasma membrane. It was hypothesized that S-acylation in
concert with binding to cholesterol target M2 to the edge of
the viral budding site [40], a large coalesced raft phase that is
organized by HA [30,31]. Complete immersion of the protein
in the more ordered, hence thicker, raft domains is thought to
be prevented by the relatively short TMR of M2. Thereby M2
becomes located in an ideal position to mediate the scission
of virus particles, supposedly triggered by the amphiphilic
helix in the cytoplasmic tail of M2, which is inserted into
the membrane in a wedge-like manner to induce curvature
[42,43].
We recently demonstrated that the cytoplasmic tail of M2
binds to membranes, both in vitro and in cells, and that the
membrane-binding properties are modulated by exchange
of the acylation site and of two tyrosine residues of the
CRAC motif, which mediate cholesterol binding [44]. Thus
one biochemical requirement of this model, association of
the amphiphilic helix with membranes to (possibly) induce
curvature is fulfilled.
Mixed results were obtained when raft localization of
M2 was tested experimentally. M2 expressed in transfected
cells is not associated with DRMs [33] and M2 does not
interact with the double-acylated marker for inner leaflet
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Biochemical Society Transactions (2013) Volume 41, part 1
rafts in FRET experiments [45]. However, in similar FRET
experiments M2 associates with HA [45], which is present in
a large raft domain. Likewise, using immuno-EM (electron
microscopy), M2 has been localized to the base of budding
filamentous virus particles [43]. In addition, preparation of
giant plasma membrane vesicles from cells expressing M2–
GFP (green fluorescent protein) showed that the protein is
partly present in the coalesced raft phase. Raft-targeting of
M2 does not require the CRAC motifs, but is dependent on
palmitoylation, similar to cellular proteins that were tested
with this model system [27,44]. Although localization of M2
to the edge of a raft could not be directly demonstrated in
any experiments, the results suggest that M2 has features of
both a raft-associated and a non-raft-associated protein.
Does palmitoylation and cholesterol-binding affect the
various activities of M2? Loss of the palmitoylation site
does not influence the ion channel activity of M2 [46].
More surprisingly, acylation does not affect the production
of virus particles either. In cell culture, recombinant viruses
where the acylated cysteine in M2 (or in CM2 of influenza
C virus) has been replaced grow as similarly well as the
corresponding wild-type virus [41,47], even if acylation is
deleted simultaneously with the cholesterol-binding CRAC
motif [48]. Moreover, 15 % of sequences of natural virus
strains present in the NCBI database lack the acylation
site in M2 [48]. Intriguingly, however, attenuation of virus
infectivity is observed on infection of mice with virus
containing non-acylated [49] or CRAC-disrupted [50] M2.
This indicates that there is a more complex influence of M2
in the context of the infected host that is not accounted for in
cell culture experiments.
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Funding
The joint work carried out in the laboratory of M.V., M.V.S. and
L.V.K. is funded by the German Research Foundation [grant number
Ve 141/10] and by the Russian Foundation for Basic Research
[grant numbers 10-04-91333 and 12-04-01695]. Other work in
the laboratory of M.V. is funded by the DFG (Priority Programme
1175, Collaborative Research Centre 740) and by 7th Framework
Programme of the European Commission (Marie Curie Initial Training
Network ‘Virus-Entry’).
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Received 20 August 2012
doi:10.1042/BST20120210
C The
C 2013 Biochemical Society
Authors Journal compilation 55