Download Measles virus M and F proteins associate with detergent

Journal of General Virology (2007), 88, 1243–1250
DOI 10.1099/vir.0.82578-0
Measles virus M and F proteins associate with
detergent-resistant membrane fractions and
promote formation of virus-like particles
Christine Pohl,1 W. Paul Duprex,2 Georg Krohne,3 Bertus K. Rima2
and Sibylle Schneider-Schaulies1
Correspondence
1
Sibylle Schneider-Schaulies
[email protected]
2
Institute for Virology and Immunobiology, University of Wuerzburg, Versbacher Str. 7,
D-97078 Wuerzburg, Germany
Centre for Cancer Research and Cell Biology, School of Biomedical Sciences,
The Queen’s University of Belfast, Belfast BT9 7BL, Northern Ireland, UK
3
Department for Electron Microscopy, University Wuerzburg, Biocenter, D-97078 Wuerzburg,
Germany
Received 20 September 2006
Accepted 11 December 2006
Assembly and release of particles comprise a late step in virus–host cell interactions. Though it
may share major biological properties with its orthologues in related viruses, trafficking and
oligomerization of the matrix (M) protein of Measles virus (MV) and its relative contribution to
assembly and budding of particles from particular host cells have not been addressed in more
detail. Plasmid-driven expression of authentic and mutant M proteins revealed that the amino acid
at position 89, an important adaptation determinant for growth of attenuated strains in Vero cells,
influences the electrophoretic mobility but not the intracellular distribution of M proteins, nor
their ability to oligomerize or migrate as a doublet band in SDS-PAGE. M proteins were found to
co-float with detergent-resistant membrane fractions (DRM) and this was enhanced upon
co-expression of the F protein. In contrast to their DRM association, the ability of M proteins to
promote release of virus-like particles (VLPs) was not affected by the presence of F proteins,
which on their own also efficiently promoted VLP production. Thus, DRM recruitment of MV F and
M proteins and their ability to drive particle formation are not correlated.
INTRODUCTION
Measles virus (MV) causes an acute, self-limiting infection
which is associated with high morbidity/mortality rates
worldwide due to its ability to induce a profound, transient
immunosuppression (Clements & Cutts, 1995). Both its
receptor usage and its ability to replicate in particular
host cells are important in MV pathogenesis (SchneiderSchaulies et al., 2001; Vincent et al., 2002; Yanagi et al.,
2002). Whilst MV replicates efficiently in many cell types
originating from humans or non-human primates, particular restrictions in these and in most rodent cells have
been described. These can arise spontaneously or depend
on cellular differentiation and occur at various levels, also
including viral budding and/or production of infectious
virus particles (Helin et al., 1999; Niewiesk et al., 1997;
Schneider-Schaulies et al., 1993, 1995; Vincent et al., 1999,
2002).
As a member of the order Mononegavirales, MV contains a
negative-stranded non-segmented RNA genome tightly
encapsidated by nucleocapsid (N) proteins and associated
with the viral polymerase complex, consisting of the large
(L) and phospho (P) proteins (Horikami & Moyer, 1995;
0008-2578 G 2007 SGM
Rima, 1996). The ribonucleoprotein complex is surrounded by a host cell-derived lipid bilayer from which
two glycoproteins project. The haemagglutinin (HA)
protein binds to the cellular receptors CD46 and/or
CD150 (Dorig et al., 1993; Naniche et al., 1993; Tatsuo
et al., 2000), and the proteolytically activated, disulphide
bridge-linked fusion (F) protein mediates membrane
fusion at neutral pH (Wild & Buckland, 1995). The
interaction of the cytoplasmic tail of the MV F protein with
the matrix (M) protein has been documented both
physically and biologically (Cathomen et al., 1998b; Moll
et al., 2002; Naim et al., 2000). In infected or transfected
cells, the M protein has an intrinsic property to associate
with cellular membranes, probably through its hydrophobic surface (Manie et al., 2000; Riedl et al., 2002). Both in
infected and transfected cells, the M protein associates with
detergent-resistant membrane (DRM) fractions, which are
thought to provide platforms for assembly and budding
(Manie et al., 2000; Vincent et al., 2000). Apparently,
neither budding from these sites nor the cooperation of M
protein with MV glycoproteins are essential since infectious particles are produced from recombinant MVs
expressing vesicular stomatitis virus (VSV) G protein
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C. Pohl and others
instead of the MV glycoproteins (Spielhofer et al., 1998).
Interestingly, MV budding does not fully rely on M protein
expression either, as infectious MV can also be released in
the absence of this protein (Cathomen et al., 1998a).
However, this only occurs at very low levels and the nature
of the infectious particle released is not clear. This suggests
that the M protein, similarly to its orthologues in related
viruses, is a driving force for MV budding (Hartlieb &
Weissenhorn, 2006; Timmins et al., 2004). Many mutated
M proteins have been described and this has been
considered as important for the lack of particle formation
in and maintenance of persistent infections (reviewed by
Billeter et al., 1994). In order to evaluate its function in MV
budding, we have studied the ability of M protein to drive
formation of virus-like particles (VLP). We noted that the
amino acid residue at position 89, which is known to be an
important determinant for MV growth in Vero cells
(Miyajima et al., 2004; Tahara et al., 2005), affects the
electrophoretic mobility of this protein. M proteins were
found to oligomerize and to partially partition into DRM
fractions. We found that both MV M (a fraction of which
is modified by ubiquitination) and F proteins individually
promote the formation of VLPs, although they do not act
in synergy. Thus we propose that M and F act separately in
MV particle morphogenesis and release.
METHODS
Cells and viruses. HeLa and 293 cells were maintained in minimal
essential medium (MEM) containing 5 % or 10 % fetal calf serum
(FCS), respectively, and 293 cells transfected to stably express the
Edmonston (ED)- or wild-type (WT)-derived F protein [293-F(ED)
and 293-F(WTF)] were maintained in MEM 10 % FCS supplemented
with 0.5 mg G418 ml21. MV vaccine strain Edmonston (ED) was
grown in Vero cells in MEM 5 % FCS and titrated on marmoset
lymphoblastoid B95a cells (maintained in RPMI 1640 supplemented
with 5 % FCS). Following infection of HeLa or 293 cells, cell media
were supplemented with 0.2 mM fusion inhibitory peptide (FIP) (Z)D-Phe–L-Phe–Gly-OH (Bachem) in order to prevent membrane
fusion and viral spread.
Indirect immunofluorescence, flow cytometry and transmission
electron microscopy (TEM). For indirect immunofluorescence, cells
were fixed in 4 % (w/v) paraformaldehyde in PBS and stained using a
mouse monoclonal a-M antibody (MAB8910; Chemicon) followed by
a goat a-mouse antibody conjugated to Alexa Fluor 594 (Molecular
Probes). For flow cytometry, fixed cells were permeabilized in PBS
containing 0.5 % BSA, 0.33 % saponin, 0.02 % NaN3, pH 7.4 and
incubated with MAB8910 or B347, a monoclonal antibody generated
in our laboratory after immunization of mice with MV ED and an
FITC-conjugated goat a-mouse antibody (Dianova), prior to analysis
by flow cytometry in a FACSCalibur system using CellQuest Pro
software (both BD Biosciences). A VSV G-specific antibody, kindly
provided by Matthias Schnell (Thomas Jefferson University,
Philadelphia, PA, USA), served as isotype control. For TEM, cells
were fixed in 0.05 M cacodylate buffer containing 2.5 % glutaraldehyde (pH 7.2), dehydrated, embedded in Epon and ultrathinsectioned. For electron microscope immunolocalization, cells were
fixed using 4 % paraformaldehyde, treated with 50 mM NH4Cl,
dehydrated and embedded in LR-White. Ultrathin sections were
incubated with MAB8910 and a secondary goat a-mouse antibody
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conjugated to 12 nm colloidal gold (Dianova), and analysed with an
EM10 transmission electron microscope (LEO, now Zeiss).
Plasmid constructions, transfections and detection of proteins.
The plasmids used were pCG-M(ED), pCG-M(ED89KAE), pCGM(WTF) and pCG-M(WTF89EAK). They were generated in
pCG(DC), a modified eukaryotic expression vector which contains
the cytomegalovirus immediate-early promoter and DraI and CspI
restriction sites. These sites facilitate the directional cloning of fulllength MV M genes which were amplified by RT-PCR from viral
RNA or by PCR from existing clones using primers priMVuniM+
(59-GTCAGGTTTAAAGTGATTGCCTCCCAAGTTC-39) and priMVuniM2 (59-TCACCTCGGTCCGTTGTGCGGTTCGGTTGTGG39) containing equivalent restriction sites (underlined). Site-directed
mutagenesis using a QuikChange kit (Stratagene) was used to
introduce an AAG point mutation into pCG-M(ED) to generate
pCG-M(ED89KAE) and a GAA point mutation into pCG-M(WTF)
to produce pCG-M(WTF89EAK). M proteins were expressed by
transient transfection into HeLa cells using Superfect reagent
(Qiagen) or 293, 293-F(ED) and 293-F(WTF) cells using Saint-Mix
(Synvolux) according to the manufacturer’s instructions. Lysates were
prepared according to standard procedures and analysed by SDSPAGE (15 % gel). For separation of the M-specific bands into
doublets, long-distance separation, low current and low temperature
conditions were used, while separated bands could be shifted back
into one by short-run and short-distance electrophoresis. For
immunoblot analysis, MAB8910 and a horseradish peroxidaseconjugated goat a-mouse antibody (Dianova) (for detection of M
proteins) or a polyclonal rabbit antiserum raised against the
cytoplasmic tail of F protein followed by a horseradish peroxidaseconjugated goat a-rabbit antibody (Cell Signaling) (for detection of
the MV F protein) were used. Secondary conjugates were detected by
using enhanced chemiluminescence (ECL) reagent (Amersham) and,
when indicated, signals were quantified by using AIDA software
(Raytest).
Co-immunoprecipitation. Plasmid pBJ5-HA-Ub (which encodes
ubiquitin fused with the influenza virus HA epitope tag, kindly
provided by Dirk Lindemann, Technical University Dresden,
Germany) (Strack et al., 2002) was either singly or co-transfected
with pCG-M(ED) into HeLa cells. Prior to lysis, cells were treated
with MG132 (10 mM in DMSO for 4 h), and lysed 48 h posttransfection in RIPA buffer [0.15 M NaCl, 1 % NP40, 0.5 % sodium
deoxycholate, 0.1 % SDS, 0.05 M Tris (pH 8.0), EDTA-free protease
inhibitor cocktail (Roche), 5 mg pepstatin ml21] supplemented with
10 mM N-ethylmaleimide (Sigma-Aldrich). Immune complexes,
obtained after precipitation using monoclonal antibody F-7 which
is specific for the HA epitope tag (Santa Cruz Biotechnology), were
analysed by SDS-PAGE (15 % gel) and M protein was detected by
immunoblotting.
Floatation assays. Cells were lysed in cold lysis buffer containing
0.1 % Brij 98 in NTE buffer [25 mM Tris (pH 7.5), 150 mM NaCl,
5 mM EDTA, 1 mM NaF] and EDTA-free protease inhibitor cocktail
(Roche) for 20 min on ice. After mixing with 3 vols 60 % sucrose in
NTE buffer, lysates were overlaid with 30 % sucrose in NTE buffer
(12.5 vols) and 2.5 % sucrose in NTE buffer (4 vols). Samples were
centrifuged at 300 000 g for 16 h at 4 uC. Fractions were collected from
the bottom to the top of the gradient and proteins were precipitated
with 2 vols cold acetone, and analysed by SDS-PAGE (15 % gel)
followed by immunoblotting. DRM fractions were detected using H319, a rabbit polyclonal serum raised against CD55 (Santa Cruz
Biotechnology) followed by a secondary goat-a-rabbit antibody (Cell
Signaling).
Detection of viral and virus-like particles. Supernatants were
collected 48 h post-transfection or -infection of 293 or HeLa cells
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Journal of General Virology 88
Measles virus envelope proteins and particle formation
(36106 or 7.56105 cells, respectively) and subjected to low-speed
centrifugation (10 min, 4 uC at 3000 g) followed by ultracentrifugation
through a 20 % sucrose cushion [in 10 mM Tris (pH 7.4), 0.1 M NaCl,
1 mM EDTA] for 2 h, 4 uC at 150 000 g. Pelleted material was
resuspended in SDS sample buffer. Cell lysates were also obtained
from the cultures and 1/60 of the total lysate was subjected, together
with the pelleted material, to SDS-PAGE (15 % gel) followed by
immunoblotting to detect F or M proteins. Relative accumulation
levels of M or F proteins in particles were calculated after determining
the total amount of the respective protein in each lysate and its
corresponding supernatant (together set to 100 %) using AIDA
software (Raytest).
RESULTS
The amino acid at position 89 affects
electrophoretic mobility but not subcellular
distribution or oligomerization of MV WT- and
vaccine strain-derived M proteins
Exchange of the conserved asparagine (E) residue in MV
WT strains for lysine (K) (found in attenuated MV strains)
was recently found to be important for MV growth on
Vero cells (Miyajima et al., 2004; Tahara et al., 2005). To
establish whether exchange of this residue affects the
general biological properties of M proteins, we generated
pCG-based constructs to drive expression of the authentic
[pCG-M(ED) and pCG-M(WTF)] and point-mutated
[pCG-M(ED89KAE) and pCG-M(WTF89EAK)] M proteins. When transfected into HeLa cells, all constructs gave
rise to proteins detectable by two monoclonal M proteinspecific antibodies (MAB8910 and B347) by flow cytometry
(Fig. 1a). Indirect immunofluorescence analysis revealed
that they did not differ with regard to their intracellular
distribution and localized mainly to the cytosol, but also to
the plasma membrane (Fig. 1b). Next we tested whether aa
89 affected the ability of MV M proteins to oligomerize,
which has been found important for membrane trafficking
and budding in related viruses (Gomis-Ruth et al., 2003;
Panchal et al., 2003; Timmins et al., 2003). Immunoblot
analysis performed with transfected HeLa cell extracts,
which were left unboiled prior to SDS-PAGE separation,
revealed that all M proteins were able to oligomerize at
similar efficiencies (Fig. 1c). As particularly visible when
monomers are expressed at high levels [M(ED89KAE) and
M(WTF)], M proteins did not migrate as discrete bands,
indicating they might carry a post-translational modification (Fig. 1c, and see below). When our standard SDSPAGE conditions were used for separation, M proteins
detected in transfected and ED-infected HeLa or 293 cells
reproducibly migrated as double bands, irrespective of the
amino acid at position 89. Exchange of E for K at this
position affected the overall electrophoretic migration
pattern of these proteins (Fig. 1d). Thus, 89KAE shifted
M(ED) to a higher and 89EAK M(WTF) to a lower
Fig. 1. Amino acid 89 influences the electrophoretic mobility but not the subcellular
distribution and oligomerization of MV M
proteins. Expression of M(ED), M(WTF),
M(ED89KAE) and M(WTF89EAK) was
detected 24 h after transfection of the corresponding expression constructs into HeLa
cells by flow cytometry using MAB8910 (red
lines) or B347 (blue lines) with a mouse
isotype IgG (black lines) serving as control
(a), by indirect immunofluorescence (magnification: 40¾) (b) or by immunoblot in extracts
left unboiled prior to SDS-PAGE (c). Extracts
were prepared 24 h following transfection of
HeLa or 293 cells [with lysates of ED-infected
(ED) or uninfected (co) cells used as controls]
and analysed by immunoblot using standard
separation conditions (d).
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C. Pohl and others
apparent molecular mass (by about 2 kDa) than the
authentic proteins [M(ED), 37 kDa and M(WTF),
39 kDa)]. Apparently, the established difference in electrophoretic migration for M proteins derived from attenuated
and wild-type MV strains (Rima, 1983; Rima et al., 1979,
1995) is largely, but not entirely, determined by aa 89
since the mutant M proteins migrated similarly, but not
identically, to the authentic proteins (Fig. 1d). Since, apart
from the overall migration pattern, aa 89 had no detectable
impact on the biological properties of M proteins assayed
so far, we confined all subsequent analyses to the authentic
M(ED) and M(WTF) proteins.
MV M protein associates with DRM fractions and
this is enhanced upon co-expression of F protein
In HeLa cells infected with MV or recombinant vaccinia
viruses encoding MV proteins, association of a fraction of
M and F proteins with and preferential budding of virus
from DRM domains containing the glycosylphosphatidylinositol (GPI)-anchored CD55 have been described (Manie
et al., 2000; Vincent et al., 2000). In agreement with these
studies, we observed that approximately 10 % of the total
M protein pool associates with CD55-containing DRM
fractions obtained from extracts of ED-infected or pCG-Mtransfected 293 cells [Fig. 2a, b; shown for ED(M)]. In
agreement with previous findings in HeLa cells, the
majority of the F protein was also found to co-float with
CD55 in 293 cells stably or transiently expressing this
protein (not shown). When our standard separating
conditions were used (Fig. 2a, b, d), M-specific doublets
Fig. 2. MV M protein associates with detergent-resistant membrane fractions and this is enhanced in the presence of F protein.
Lysates prepared from 293 cells infected with ED (m.o.i. 0.5, 24 h)
(a) or 48 h post-transfection of pCG-M(ED) into 293 cells (b),
293-F(ED) cells (c) and 293-F(WTF) cells (d) were subjected to
sucrose-gradient centrifugation. Fractions were analysed for the
presence of M protein and CD55 (a GPI-anchored protein,
commonly used as DRM marker) by immunoblotting (representatively shown in the bottom panel). In (c), F protein was co-detected
using an F-specific serum.
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equivalent to those described (Fig. 1d) were also detectable,
yet neither of these protein species associated preferentially
with the DRM or the detergent-soluble fractions. To
investigate whether co-expression of F protein would alter
DRM association of M proteins, pCG-M(ED) was transfected into 293 cells stably expressing authentic (EDderived) (Fig. 2c) or heterologous (WTF-derived) (Fig. 2d)
F proteins. Though accumulation levels of M protein in both
stable transfectant cell lines were lower than in parental 293
cells, co-expression of either F protein detectably enhanced
the levels of DRM-associated M protein, in contrast to what
has been described in vaccinia-driven expression systems
(Vincent et al., 2000). This increase was about fourfold as
determined after quantification of the percentage of total M
protein amounts in the respective extracts, thus indicating
that the presence of F protein allows M protein accumulation at these sites.
M and F proteins promote, but do not cooperate
in, formation of VLPs
The ability of MV M and/or F proteins to promote particle
formation was analysed directly by TEM on ED-infected
and pCG-M(ED)-transfected 293 or 293-F(ED) cells.
Vesicular structures budding from the surface were clearly
detectable in 293 and 293-F(ED) cells transfected to express
M(ED) protein and in 293-F(ED) cells alone (Fig. 3b–e,
upper row), but not in untransfected cells or cells
transfected to express N protein (Fig. 3a and not shown).
VLPs released after expression of M or F proteins alone or
in combination tended to be smaller than viral particles
(Fig. 3b–e). Particles released from MV-infected (Fig. 3g)
and pCG-M(ED)-transfected 293 (Fig. 3h) and 293-F(ED)
cells (Fig. 3i) did contain M protein, as confirmed by
MAB8910 binding followed by detection by a secondary
antibody conjugated to colloidal gold particles. The ability
of M protein orthologues to promote VLP formation is
often linked to transient mono-ubiquitination of these
proteins (Hartlieb & Weissenhorn, 2006; Martin-Serrano et
al., 2005). To investigate whether at least a fraction of MV
M proteins carries this modification, 293 cells were cotransfected with pCG-M(ED) and pBJ5-HA-Ub, from
which HA-tagged ubiquitin (HA-Ub) is expressed, and
exposed to a proteasome inhibitor, MG132, prior to extract
preparation to stabilize potentially ubiquitin-modified
proteins. Indeed, M(ED) protein was precipitated by the
HA-specific antibody from lysates of doubly transfected
cells. The only marginally slower electrophoretic mobility
than that seen for the majority of M protein in the cell
lysate suggests that it is most likely mono-ubiquitinated
(Fig. 4a).
To assess VLP formation driven by M proteins on a
quantitative basis, their relative amounts in supernatants of
293 or HeLa cells transfected with pCG-M(ED) or pCGM(WTF) were determined. They comprised, on average,
up to 2 % of the total M protein pools of the transfected
cultures (Fig. 4b, d). This percentage did not differ between
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Measles virus envelope proteins and particle formation
Fig. 3. (a–e) TEM analysis of particle release from 293 and 293-F(ED) cells transfected to express M protein 48 h postinfection or post-transfection. (a) Uninfected, (b) ED-infected and (c) pCG-M(ED)-transfected 293 cells and (d) pCG-M(ED)transfected 293-F(ED) cells. (f–i) Detection of M protein by immunogold labelling in (f) uninfected, (g) ED-infected and (h)
pCG-M(ED)-transfected 293 cells and (i) pCG-M(ED)-transfected 293-F(ED) cells. Positions of some gold particles are
indicated by arrows (magnification: 25 000¾).
M(ED) and M(WTF) [Fig. 4d, and not shown for
M(WTF)]. Accumulation levels of M protein in supernatants of infected 293 cells were almost identical to those
determined for M-driven VLPs, confirming that particle
production by MV is very inefficient (Fig. 4d). Notably, the
M protein released separated into doublets (Fig. 4b, c).
However, in contrast to its association with DRM fractions
(Fig. 2c), accumulation of M protein in supernatants was
not enhanced by the presence of F protein provided by
293-F(ED) cells (Fig. 4d). This was also seen upon transient
co-expression of either ED- or WTF-derived F protein and
in 293-F(WTF) cells [which stably expresses the F(WTF)
protein], indicating that there is no obvious correlation
between M protein-driven VLP production and the
amount of protein associated with DRMs (not shown).
Surprisingly, F protein was found to be released from 293-F
cells rather efficiently (Fig. 4c, upper panel), accumulation
levels comprising on average 6 % of the total F protein
pools, independently of stable or transient expression of F
protein or of co-expressed M protein (Fig. 4c, and not
shown). Thus, the M and F proteins seem not to act
cooperatively in driving VLP production (Fig. 4d). Again, the
amount of F proteins pelleted from supernatants of infected
cells corresponded to that seen for the VLPs (Fig. 4d).
DISCUSSION
MV M proteins have been studied in detail since, about
two decades ago, they were found to be extensively
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mutated in persistently infected brain cells. Such mutations
were shown to cause aberrant expression, functional
impairment, instability or complete absence of these
proteins (reviewed by Rima & Duprex, 2005; SchneiderSchaulies et al.,1995, 2003). Although abrogation of M
protein expression or function has been considered as
causatively linked to the lack of virus production in the
central nervous system, the role of this protein in
promoting particle assembly and release has not been
satisfactorily addressed.
M proteins of vaccine and WT strains have long been
known to differ with regard to their electrophoretic
mobility in SDS-PAGE (Rima et al., 1995, 1979). We have
now established that the amino acid residue at position 89
largely accounts for this phenomenon (Fig. 1d) and that,
although other residues distinctive for WT strains (such as
aa 64 and aa 209) may also play a part, their contribution is
much less significant. Substitution of E by K at aa 89 was
recently shown to greatly increase the ability of a WT
recombinant MV to replicate in Vero cells, as did
substitution of aa 64 (PAS) (Miyajima et al., 2004;
Tahara et al., 2005). However, the consequences of these
particular substitutions in functional terms have not been
further addressed in these studies and thus remain to be
established. Clearly aa 89 does not detectably affect the
biological properties of M proteins further, for example,
their ability to oligomerize (Fig. 1c) and thus, similarly to
its functional analogue Ebola virus VP40 protein, is likely
to be of critical importance in driving particle formation
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C. Pohl and others
Fig. 4. MV M and F protein promote release of
virus-like particles. (a) M protein was detected
in whole-cell lysates (CL) or a-HA precipitates
(IP) of lysates prepared from HeLa cells
transfected to express HA-tagged ubiquitin
(HA-Ub) alone or in combination with M(ED)
and treated with MG132 4 h prior to lysis. (b)
Supernatants (SN) of 293 cells either left
untreated (co), ED-infected [m.o.i. 0.2, 48 h,
(ED)], pCG-M(ED)-transfected, or pCGM(WTF)-transfected (upper panel). HeLa cells
transfected with pCG-M(ED) or pCG-M(WTF)
(bottom panel) for 48 h were subjected to
ultracentrifugation and their supernatant and
lysate were analysed for the presence of M
protein by immunoblot analysis (upper panel).
(c) Samples obtained from ED-infected and
pCG-M(ED)-transfected 293 cells, and from
untransfected and pCG-M(ED)-transfected
293-F(ED) cells were analysed as described
in (b) for the expression of M and F proteins.
(d) The percentage of M (black bars) and F
proteins (grey bars) released into the supernatant from ED-infected 293 cells and 293F(ED) cells transiently expressing M(ED) was
determined by using AIDA software. The amount
of protein released into the supernatant and
present in the cell lysate was recalculated and
set to 100 %. (M, n54; F, n53).
(Gomis-Ruth et al., 2003; Hoenen et al., 2005; Panchal
et al., 2003). Although M proteins transiently expressed in
Madin–Darby canine kidney (MDCK) cells did not
associate with the plasma membrane (Riedl et al., 2002),
a substantial fraction of all M proteins analysed in our
study were readily detectable on the cell surface (Fig. 1b).
Since MV M proteins contain all of the signals for budding,
as is evident from their ability to promote VLP formation
independently (Figs 3, 4), they would be expected to
accumulate at the cell membrane. Thus, the failure of M
proteins to associate with the plasma membrane in MDCK
cells might relate to the polarized phenotype of these cells,
which could influence protein trafficking. It might be
interesting to determine whether this affects VLP production as well.
We have shown that M proteins can reproducibly be
separated into doublets (Figs 1, 2, 4) and that this is
independent of both their origin and the specific amino
acid at position 89. This critically depended on the SDSPAGE conditions and the doublet could be converted into
a single band when acrylamide concentration, separation
distance or running conditions were altered, for example,
when we modified the separation distance deliberately to
enable documentation of the ubiquitinated M protein
fraction (Fig. 4a). Although it is tempting to speculate that
the two M protein species detected arose from differential
post-translational modifications, it is unclear what these
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could be. Lipidation, as typically observed for membraneassociated proteins, including human immunodeficiency
virus gag protein, would be the most likely posttranslational modification. As yet, only F protein has been
found to be palmitoylated in MV-infected cells (Caballero
et al., 1998). Although they cannot be excluded, modification by phosphorylation or ubiquitin conjugation is
unlikely to account for the two M protein species; the first
has never been evidenced for MV M protein, and the ease
of detection of the doublet as compared to the difficulty to
detect the minor fraction of ubiquitinated M proteins only
after addition of MG132 (Fig. 4a) argues against the second
one.
As seen for their orthologues, MV M proteins are
apparently transiently mono-ubiquitinated (Fig. 4a) and
this can only be detected for a minor fraction of this protein,
due to the highly transient nature of this modification
(Demirov & Freed, 2004; Hartlieb & Weissenhorn, 2006;
Martin-Serrano et al., 2005, 2004; Strack et al., 2000). The
latter is consistent with the requirement of this class E
factor-dependent modification for initiation of sequential
recruitment of ESCRT (endosomal sorting complex
required for transport) complexes and subsequent sorting
and vesicle budding into late endosomal compartments
(also referred to as multivesicular bodies) or, for viruses,
sorting to and ‘pinching off’ from plasma membranes.
Whether M protein trafficking and/or budding of MV VLPs
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Journal of General Virology 88
Measles virus envelope proteins and particle formation
or entire particles indeed involves a retrograde endosomal
transport, as shown for Ebola virus VP40, needs to be further
investigated (Kolesnikova et al., 2004a, b). Classical L
domains, which are known to be crucial for recruitment of
ESCRT complexes and subsequent sorting in retroviral gag
and M proteins of filo- and rhabdoviruses (Freed, 2002;
Timmins et al., 2004), have not as yet been identified in MV
M proteins. Our finding that M proteins promote VLP
formation suggests the presence of at least one functional L
domain; the highly conserved YMFL (aa 52–55) or PSVP (aa
311–314) motifs could be candidates for interaction with
ALIX/AIP-1 or Tsg101, respectively. A recently described L
domain for paramyxoviruses (FPIV) has not been detected
within the MV M protein sequence (Schmitt et al., 2005).
formation (Takimoto et al., 2001). However, this is not
contained within the C terminus of MV F protein. Thus,
both domains are important for this activity and its
mechanism needs to be addressed in future experiments.
Remarkably, the M proteins, but even more frequently, the
cytoplasmic tails of F proteins are loaded with mutations in
persistent MV brain infections. The experimental setups
established in our manuscript enable, amongst other
follow-up studies, to evaluate to what extent these
mutations might restrict particle production and thus
contribute to establishment of persistence.
Consistent with earlier findings (Manie et al., 2000;
Vincent et al., 2000), a fraction of the total individually
expressed M and F proteins co-floated with DRMs (Fig. 2).
Whilst in these studies F protein failed to recruit M protein
into DRMs, we reproducibly observed about a fourfold
increase in DRM-resident M protein upon stable or
transient co-expression of either an autologous or heterologous F protein (Fig. 2c, d). The extent to which the
cytoplasmic tail of F protein contributes to this recruitment
could not be determined at present, since the only antibody
able to detect this protein by immunoblot is directed
against its cytoplasmic tail and thus does not recognize F
proteins lacking this particular domain. The reasons for the
differences in the study by Manie et al. (2000) and ours are
unclear. They may, however, reflect the use of different
expression systems. Although M and F proteins apparently
cooperate in terms of DRM recruitment, they do not with
regard to VLP formation. Whilst both proteins can
promote VLP formation on their own, the amount of M
or F protein, respectively, released from co-transfected
cultures does not significantly increase, and, remarkably,
corresponds almost exactly to that seen in virions (Fig. 2c).
Firstly, this indicates that the ability of MV proteins to
promote particle formation and their DRM association are
not correlated; this may be influenced by the fact that
DRMs isolated by standard procedures contain both
plasma and internal membrane fractions, while particle
formation should be confined to plasma membraneassociated proteins only. However, it is clear that MV
budding is not strictly confined to DRMs (Vincent et al.,
2000). Secondly, in contrast to Ebola virus where
glycoprotein co-expression markedly enhances VP40driven particle production (Licata et al., 2004), MV F fails
to enhance M protein-driven VLP formation. Due to a
current lack of reagents it was not possible to determine
whether VLPs released from doubly transfected cells
contained M or F proteins alone or both. Similar to their
orthologues in MV, simian virus 5 M and F proteins can
induce budding which results in the production of VLPs
containing M and F proteins (Takimoto & Portner, 2004).
A sequence motif (TYTLE), conserved within the cytoplasmic domains of Sendai virus and human parainfluenza
virus 1 F proteins, was found to be required for particle
We thank Jürgen Schneider-Schaulies and Elita Avota for critical
reading of the manuscript and helpful comments, Mareike Berning,
Maren Klett, Elisabeth Meyer-Natus and Beatrix Loth for technical
help, and the Deutsche Forschungsgemeinschaft and Medical
Research Council (Grant 9901004) for financial support of this work.
http://vir.sgmjournals.org
ACKNOWLEDGEMENTS
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