bl
in
ch-S
L),
Received 31 August 2005; accepted 10 September 2005
bola and Marburg
is the main driving
all viral structural
nce, assembly and
ents are assembled
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
unknown natural host (Feldmann et al., 2003, 2004). Filo-
viruses contain a single-stranded negative-sense RNA genome,
VP24 are unique to the Filoviridae (Feldmann and Kiley,
1999). Although all of the structural information to build a viral
uge body of recent
enveloped viruses
Virology 344 (2006)which displays a genome organization similar to the otherIntroduction
Filoviruses are the causative agent of a severe, mostly fatal
hemorraghic fever in humans. The route of transmission
involves direct contact with body fluids from either infected
patients or non-human primates or contact with a to date
family members of the order Mononegavirales such as
Paramyxoviridae, Rhabdoviridae and Bornaviridae. The 19-
kb large filovirus genome encodes the structural proteins NP
(major nucleoprotein), VP35 (P-like protein), VP40 (matrix
protein), GP (glycoprotein) VP30 (minor nucleoprotein), VP24
and L (RNA-dependent RNA polymerase). Both VP30 andD 2005 Elsevier Inc. All rights reserved.
Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Different conformations of the matrix protein VP40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Cellular factors implicated in enveloped virus budding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
VP40 interaction with cellular factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Cellular transport of VP40 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
The role of the glycoprotein GP in assembly and budding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Assembly of the nucleocapsid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Abstract
Filoviruses belong to the order of negative-stranded non-segmented RNA viruses and are classified into two genera, E
viruses. They have a characteristic filamentous shape, which is largely determined by the matrix protein VP40. Although VP40
force for assembly and budding from the host cell, the production of infectious virus involves an intricate interplay between
proteins in addition to cellular factors, e.g., those that normally function in multi-vesicular body biogenesis. As a conseque
budding steps are defined to specific cellular compartments, and the recent progress in understanding how the different compon
into stable enveloped virus particles is reviewed.Filovirus assem
Bettina Hartlieb a,b, W
a Institut fur Virologie, Robert-Kob European Molecular Biology Laboratory (EMB0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2005.09.018
* Corresponding author. Fax: +33 476 207199.
E-mail address: [email protected] (W. Weissenhorn).y and budding
fried Weissenhorn b,*
tr. 17, 35037 Marburg, Germany
6 rue Jules Horowitz, 38042 Grenoble, France
64 70
www.elsevier.com/locate/yviroparticle is encoded in the viral genome, a h
work has shown that filoviruses like othersuch as HIV-1 hijack cellular protein machines in order to
mediate assembly at and budding from cellular membranes
(Morita and Sundquist, 2004; Schmitt and Lamb, 2004).
/ VDifferent conformations of the matrix protein VP40
The matrix protein VP40 is the major structural protein
(Geisbert and Jahrling, 1995). Numerous studies have shown
that expression of VP40 in mammalian cells leads to the
production of virus-like particles (VLPs) with filamentous
morphology suggesting that VP40 contains the necessary
information for particle assembly and budding. However,
VP40 containing VLPs vary in diameter (40 to 80 nm),
compared to a strict diameter of 80 nm of infectious virus,indicating that other viral components such as the nucleocap-
sids determine the final particle morphology (Bavari et al.,
2002; Harty et al., 2000; Hoenen et al., 2005; Jasenosky et al.,
2001; Kolesnikova et al., 2004a; Noda et al., 2002; Swenson et
al., 2004; Timmins et al., 2001). Structural and functional
studies have shown that VP40 adopts different conformations
in vitro that have been partially linked to different functions.
VP40 is initially expressed as monomer, which folds into two
structurally related beta sandwich domains forming a closed
conformation (Dessen et al., 2000). The N-terminal domain
constitutes the oligomerization module, which allows the
formation of hexameric and octameric ring-like structures
(Ruigrok et al., 2000; Timmins et al., 2003a). The C-terminal
domain was found to be required for membrane interaction
(Jasenosky et al., 2001; Ruigrok et al., 2000; Timmins et al.,
2001). Oligomerization is achieved by the displacement of the
C-terminal domain form the N-terminal domain underlining the
metastable conformation of monomeric VP40. In vitro mem-
brane targeting experiments suggest that lipid bilayer interac-
tion may be the trigger for the conformational change
(Scianimanico et al., 2000). Electron microscopy and molec-
ular modeling indicate that hexamer formation involves the
interface, which is otherwise occupied by the C-terminal
domain in the closed conformation, which extends via a
flexible linker from the ring-like structures and thus allows
membrane interaction (Dessen et al., 2000; Nguyen et al.,
2005; Scianimanico et al., 2000; Timmins et al., 2003a). In
contrast to the hexameric form of VP40, the octameric ring-like
structure depends on the interaction with single-stranded RNA.
The octamer is formed by four anti-parallel homodimers of the
N-terminal domain that bind two RNA triribonucleotides
containing the sequence 5V-U-G-A-3V at the center of the pore.The bound RNA stabilizes the dimerdimer interactions, and
biochemical studies show that RNA interaction plays a crucial
structural role as no octamers can be formed in the absence of
RNA (Gomis-Ruth et al., 2003; Timmins et al., 2003a). It is not
known whether octameric VP40 binds viral or cellular RNA
during infection. It thus remains unclear whether RNA binding
has a sole structural role to generate octamers for a specific
function, or this function is connected to the viral RNA
metabolism. Mutagenesis studies have shown that RNA
binding and thus octamer formation is absolutely essential for
virus replication (Hoenen et al., 2005). Although VP40
octamers are efficiently incorporated into VP40 containing
B. Hartlieb, W. WeissenhornVLPs (Hoenen et al., 2005; Panchal et al., 2003), the inhibition
of octamer formation still produces VP40 containing VLPs that
are indistinguishable from VLPs containing wild type VP40(Hoenen et al., 2005). On the other hand, no VP40 octamers
can be detected in infectious Ebola virus particles by Western
blot analysis (Gomis-Ruth et al., 2003). Therefore, the precise
function of VP40 octamers stays unclear. They might play a
role in RNP formation as described for a number of matrix
proteins (Coronel et al., 2001; Kaptur et al., 1991; Schmitt et
al., 2002; Stricker et al., 1994). Such a function is in agreement
with the association of VP40 with Marburg virus RNPs present
in inclusion bodies (Kolesnikova et al., 2002). The three
different conformations of VP40 are thus prime examples of
how viral genomes with a limited capacity can encode for
protein conformations that probably exert different tasks.
However, the oligomeric or polymeric form of VP40 within
viral particles still remains elusive.
Cellular factors implicated in enveloped virus budding
HIV-1 Gag deletions and mutational analyses revealed
sequence motifs that are absolutely required for budding
(Gottlinger et al., 1991; Huang et al., 1995; Wills et al.,
1994). These motifs were termed late domains since they affect
a late step in budding (Parent et al., 1995). Since then, a
number of late domain sequences including PT/SAP, PPxY and
YP(X)nL have been described to function in both positive - and
negative-strand RNA virus assembly and budding (Morita and
Sundquist, 2004; Schmitt and Lamb, 2004). Late domains can
be functionally interchangeable, their position within the viral
protein can vary, and they can act in trans although some
context dependency is conserved (Martin-Serrano et al., 2004;
Morita and Sundquist, 2004).
Numerous recent studies show that the late domains serve as
entry points into a network of proteins that normally functions
in multi-vesicular body (MVB) biogenesis. The protein
network involved in MVB formation was first identified in
Saccharomyces cerevisiae and implicated in membrane protein
trafficking from the Golgi and plasma membranes via the
endosomal system to the lysosome for degradation. Receptor
ubiquitinylation serves as a signal for the protein transport to
endosomal membrane microdomains, which ultimately leads to
membrane invagination and vesicle budding into endosomal
structures (multi-vesicular bodies), thus delivering the cargo
from the limiting membrane of the endosome into the lumen of
the organelle. This process is mediated by non-essential
proteins known as class E Vps proteins in S. cerevisiae
(Gruenberg and Stenmark, 2004; Katzmann et al., 2002).
Briefly, initial recognition of the ubiquitinylated cargo leads
to endocytosis and interaction with class E proteins Hrs
(Vps27p) and Stam (Katzmann et al., 2003) that in turn recruits
the ESCRT-1 (Endosomal Sorting Complex Required for
Transport) complex composed of Vps23p/Tsg101, Vps28p and
Vps37p to the endosomal membrane (Bache et al., 2003;
Katzmann et al., 2001; Lu et al., 2003). In yeast ESCRT-I cargo
recognition then induces the formation of ESCRT-II (Babst et al.,
2002b), which activates the assembly of the ESCRT-III multi-
irology 344 (2006) 6470 65protein complexes that are composed of CHMP (CHarged
Multivesicular body Protein) family proteins (Babst et al.,
2002a). Finally, the activity of the AAA-type ATPase Vps4 is
/ Vrequired for disassembly of ESCRT complexes from endosomal
membranes (Babst et al., 1998, 2002a). Studies with mutant
Vps4 and CHMP fusion proteins revealed a late budding arrest
of fully assembled virus particles suggesting that ESCRT-III and
Vps4 functions are crucial for enveloped virus release (Licata et
al., 2003; Lin et al., 2005; Martin-Serrano et al., 2003; Strack et
al., 2003; von Schwedler et al., 2003).
Notably, the first identified late domain motif PTAP was
shown to bind to the cellular factor Tsg101 (ESCRT-I), which
also binds ubiquitin (Demirov et al., 2002; Garrus et al., 2001;
Sundquist et al., 2004; VerPlank et al., 2001). Similarly, a
second late domain motif, YP(X)nL mediates binding to ALIX/
AIP1 that itself interacts directly with Tsg101 via its own PTAP
motif (Strack et al., 2003; von Schwedler et al., 2003). The
third late domain motif PPxY mediates interactions with
proteins that contain WW domains, such as ubiquitin ligases
(E3 enzyme). It was long speculated that ubiquitin plays a role
in assembly and budding since ubiquitin is incorporated into
retroviral particles (Ott et al., 1998; Putterman et al., 1990),
Gag proteins are monoubiquitinylated (Ott et al., 2000), and
protease inhibitors inhibit virus budding (Patnaik et al., 2000;
Schubert et al., 2000; Strack et al., 2000). For more detailed
recent reviews on cellular factors implicated in virus assembly
and budding, see Morita and Sundquist (2004) and the article
by Bieniasz and colleagues in this issue.
VP40 interaction with cellular factors
Ebola virus VP40 contains two overlapping late domains 7-
PTAPPEY-13 while Marburg virus VP40 contains only the
putative functional, 16-PPPY-19 motif (Morita and Sundquist,
2004; Schmitt and Lamb, 2004). Ebola virus VP40was shown to
interact with Tsg101 via its PTAP motif in vivo (Martin-Serrano
et al., 2001) and in vitro with both monomeric and oligomeric
VP40 (Timmins et al., 2003b). Mammalian co-expression of
Tsg101 and VP40 indicated that VP40 recruits Tsg101 to the
plasma membrane and thus to the site of budding (Martin-
Serrano et al., 2001) which includes VP40 lipid raft localization
(Bavari et al., 2002; Panchal et al., 2003; Licata et al., 2003). In
addition, Tsg101 is incorporated into VP40 containing VLPs
(Licata et al., 2003). Tsg101 recruitment most likely includes
ESCRT-I assembly at the site of budding (Martin-Serrano et al.,
2003b; Stuchell et al., 2004; Tanzi et al., 2003) consistent with
the finding that a C-terminal truncation of Tsg101 exerts a
dominant negative effect on VP40 VLP formation (Yasuda et al.,
2003). Similarly, the downstream partners, including ESCRT-
III, are probably also recruited as a mutant Vps4 ATPase affects
VP40 VLP formation similar as in case of retrovirus budding
(Licata et al., 2003; Martin-Serrano et al., 2003a; Strack et al.,
2003; von Schwedler et al., 2003).
The importance of the second motif PPPY was first
recognized by studies that showed VP40 binding to a WW
domain from the yeast E3 ligase Rsp5 (Nedd4 homologue) and
VP40 ubiquitinylation in vitro. These experiment thus revealed
B. Hartlieb, W. Weissenhorn66a role for the late domain in VP40 release from mammalian
cells (Harty et al., 2000). The PPEY motif was since shown to
interact with WW domain 3 from human Nedd4 in vitro, whichrequires the oligomeric ring-like conformation of VP40
(Timmins et al., 2003b). This indicates that ubiquitinylation
by an E3 ligase may act on an activated conformation of VP40
that is most likely targeted to the lipid bilayer via its C-terminal
domain, which is in agreement with the plasma membrane
targeting of HECT domain containing E3 ligases via their C2
domains. Consistent with the in vitro data, dominant negative
mutants of Nedd4 inhibit budding of VP40 containing VLPs
(Yasuda et al., 2003). Recently, other HECT domain containing
ubiquitin ligases were implicated in the PPPY late domain
interaction including WWP1, WWP2 and itch (Martin-Serrano
et al., 2005). In addition, the recruitment of HECT domain
containing E3 ligases including Nedd4 into aberrant endosomal
compartments induced by mutant Vps4 provides a direct link
between the function of class E Vps factors and ubiquitinyla-
tion (Martin-Serrano et al., 2005). Such a link is also consistent
with the implication of yeast Rsp5 in ubiquitinylation of cargo
for sorting into MVB and the genetic linkage of Rsp5 and
ESCRT-III (Katzmann et al., 2004). Therefore, the presence of
a single PPxY motif in Marburg virus VP40 may be sufficient
to recruit the Vps machinery for assistance in assembly and
budding.
A number of studies have shown that the presence of intact
late domains is required for the efficient release of VP40
containing VLPs albeit some VP40 release takes place in the
absence of late domains (Harty et al., 2000; Jasenosky et al.,
2001; Timmins et al., 2001). In addition, recent work shows
that the Ebola virus VP40 late domains are not absolutely
required for virion production in cell culture, although titers of
late domain mutant virions were affected by one log unit
(Neumann et al., 2005). This thus poses the question as to
whether yet other unknown sequences in VP40 are implicated
in the assembly and budding process.
Cellular transport of VP40
Since expression of VP40 reveals mostly plasma mem-
brane targeting (Bavari et al., 2002; Harty et al., 2000;
Hoenen et al., 2005; Martin-Serrano et al., 2001; Timmins et
al., 2001), which includes lipid raft microdomain localization
(Bavari et al., 2002; Panchal et al., 2003), an active transport
of VP40 to specific cellular sites is required. In addition,
ultrastructural analyses of VP40 in Ebola virus and Marburg
virus-infected cells detect VP40 in viral inclusions and in
multi-vesicular bodies (MVBs) derived from the late endoso-
mal compartment indicating (i) an interaction of VP40 with
nucleocapsid structures and (ii) with endosomal membranes
(Geisbert and Jahrling, 1995; Kolesnikova et al., 2002,
2004a). It has been thus suggested that VP40 is transported
through the retrograde late endosomal pathway en route from
the endosome to the plasma membrane (Kolesnikova et al.,
2002, 2004a, 2004b).
The role of the glycoprotein GP in assembly and budding
irology 344 (2006) 6470The envelope of filoviruses is decorated by the trimeric
surface protein GP, a type I transmembrane protein (Feldmann
et al., 1991), which mediates cell entry (Schibli and Weissen-
horn, 2004). GP is initially expressed as a precursor protein that
is subsequently cleaved by the subtilisin-like proprotein
convertase furin in the TGN into two subunits, the receptor
binding domain GP1 and the membrane anchored fusion
protein GP2 (Volchkov et al., 1998a, 2000). Two C-terminal
cysteins between the transmembrane region and the short
cytoplasmic region are acylated (Funke et al., 1995; Ito et al.,
2001), which may play a role in a postulated interaction with
the matrix protein VP40. GP expression alone leads to its
transport to the plasma membrane via the secretory pathway
and to the release of GP containing vesicles (Kolesnikova et al.,
2004b; Sanger et al., 2001; Volchkov et al., 1998b). However,
upon co-expression of GP and VP40 alone or in Marburg virus-
infected cells, GP re-localizes to MVBs, which are also
enriched in VP40. This suggests that GP and VP40 are
transported together to the site of budding at the plasma
membrane. In addition, such a co-localization is also consistent
which are similar to those detected in Ebola and Marburg virus-
infected cells (Becker et al., 1998; Modrof et al., 2002). The
inclusion bodies contain nucleocapsid-like particles that closely
resemble the helical core of nucleocapsids incorporated into
mature virions indicating that NP determines the nucleocapsid
structure (Kolesnikova et al., 2000, 2002; Noda et al., 2005).
Although NP is the major determinant of the nucleocapsid, it
also recruits VP30, VP35 and L into the inclusion bodies
(Becker et al., 1998). In addition, VP24 and VP35 have been
previously implicated in nucleocapsid formation (Huang et al.,
2002). NP was also shown to enhance budding activity of
VP40 suggesting an NP-VP40 interaction (Licata et al., 2004).
Although VP40 interacts with NP containing nucleocapsids,
VP40 and NP do not co-localize in MVBs, indicating that two
different forms or conformations of VP40 pass by the MVB
pathway and associate with nucleocpasids (Kolesnikova et al.,
2004a, 2004b). Although it is currently not known how the
assembled nucleocapsids leave the site of inclusion bodies, it
to th
cleo
B. Hartlieb, W. Weissenhorn / Virology 344 (2006) 6470 67with virus budding into endosomal structures (Kolesnikova et
al., 2004b). Like VP40, GP is targeted to lipid raft micro-
domains in the plasma membrane, and co-expression of VP40
and GP supports and enhances the efficiency of VP40 and GP
containing VLP formation, which is morphologically indistin-
guishable from infectious Ebola virus (Bavari et al., 2002;
Noda et al., 2002). Interestingly, VLPs containing VP40 and
GP have been shown to provide protection from filovirus
infection upon VLP immunization (Warfield et al., 2003;
Warfield et al., 2004).
Assembly of the nucleocapsid
The nucleoprotein NP encapsidates the RNA genome
(Sanchez et al., 1992). Expression of NP in insect cells leads
to the formation of helical NP structures that contain cellular
RNA (Mavrakis et al., 2002). Expression of NP in mammalian
cells leads to the formation of intracellular inclusion bodies,
Fig. 1. Schematic illustration of distinct transport pathways of viral components
endosomal pathway. (B) Transport of the glycoprotein GP. (C) Transport of nuparticles containing NC, VP40 and GP. Both GP and VP40 are targeted to lipid
expression induce either the release of GP containing vesicles or VP40 containing fil
Becker (2004).might involve the cellular cytoskeleton and VP40 interactions
with microtubules (Ruthel et al., 2005). Furthermore, NP is
highly phosphorylated at its C-terminus, which might play a
role in regulating either RNAprotein or proteinprotein
interactions (Becker et al., 1994; Elliott et al., 1985).
Although VP24 has been previously implicated in nucleo-
capsid assembly (Huang et al., 2002), its function is not well
understood as nucleocapsid structures can be formed without
VP24 (Watanabe et al., 2004). In addition, Ebola virus VP24
interacts with membranes, oligomerizes and is released from
VP24 expressing cells in form of trypsin-resistant vesicles (Han
et al., 2003). Furthermore, recent studies with Marburg virus
VP24 confirmed its lipid bilayer interaction property and its
oligomerization into tetramers. However, it is also recruited
into viral inclusions containing preformed nucleocapsids as
well as into VP40 containing VLPs. Recruitment of VP24
neither influences efficiency nor morphology of released
particles (Bamberg et al., 2005). Silencing of VP24 by si-
e site of assembly and budding. (A) Transport of VP40 along the retrograde late
cpasids (NC containing NP, VP30, VP35, L and VP24) and assembly of viralraft microdomains (LR) that serve as platforms for assembly. GP and VP40
amentous VLPs (shaded areas). The figure was modified from Kolesnikova and
/ VRNA in Marburg virus-infected cells resulted in reduction of
released virions, indicating a role in assembly and/or budding,
whereas viral transcription and replication were not affected
(Bamberg et al., 2005). Accordingly, VP24 might be either
important for the assembly of transport competent nucleocap-
sids or the association of nucleocapsids with the transport
machinery or in the targeting of the nucleocapsids to the
budding sites containing GP and VP40 (Bamberg et al., 2005;
Kolesnikova et al., 2004b).
Conclusions
The matrix protein VP40 is initially expressed in a
monomeric conformation that is targeted to cellular mem-
branes. This may include recruitment of cellular factors such as
Tsg101 to the membrane or vice versa and ubiquitinylation of
membrane associated VP40 by HECT domain containing
ubiquitin ligases. Transport to the plasma membrane occurs
by the retrograde late endosomal pathway via MVBs. VP40
expression alone then leads to the targeting to lipid raft
microdomains and the release of filamentous VLPs. VP40 is
thus sufficient to re-localize the cellular budding machinery via
Tsg101 and HECT domain containing ubiquitin ligase interac-
tions to the site of virus assembly and budding. VP40 is also
found in association with viral inclusions containing assembled
nucleocapsids, probably in a conformation different from the
monomeric form such as the hexameric form or the octamers in
complex with RNA (Fig. 1A).
GP expression follows the regular secretory pathway and
leads to lipid raft microdomain localization of GP. Surprisingly,
this is sufficient for the release of GP containing vesicles (Fig.
1B). However, upon co-expression of GP and VP40 and in
Marburg virus-infected cells, GP localizes to the late endosome
after proteolytic cleavage in the TGN and accumulates in
MVBs together with VP40. Both are thus co-transported to the
site of assembly and budding (Fig. 1C). NPRNA interactions
are sufficient for nucleocapsid assembly that recruits VP30,
VP35 and L. Nucleocapsids accumulate in cellular inclusions
that co-localize with small amounts of VP40. These complexes
are then transported to VP40 and GP containing MVBs and to
the plasma membrane that leads to virus particle assembly and
release (Fig. 1C).
There are most likely a number of features common to
assembly and budding processes of enveloped viruses since
most enveloped viruses recruit cellular factors that normally
act in MVB biogenesis. Based on the available data, we
hypothesize that recruitment of cellular factors serves
potentially two major purposes. Firstly, it needs to recruit
factors that help to initiate the assembly process mediated by
the matrix protein, which probably includes initiation of
membrane curvature at the site of assembly. Such a function
may be recruited by the ubiquitinylation of matrix proteins, a
process which is linked to the endocytosis machinery, which
thus may provide such functions. Secondly, recruitment of
B. Hartlieb, W. Weissenhorn68ESCRT complexes, especially ESCRT-III and Vps4, may be
required for the last step of the budding process, the release
of the fully assembled virus particle from cellular mem-branes. Therefore, selective employment of cellular proteins
by enveloped viruses via different late domains may provide
specialized fine tuning accessories for the assembly process
and the protein machinery required for final steps in
budding.
Acknowledgments
We thank Drs. S. Becker and L. Kolesnikova for critical
reading of the manuscript. Work in the authors laboratory and
B. H.s trainee period at EMBL were supported by EMBL and
Deutsche Forschunggemeinschaft SFB 593.
References
Babst, M., Wendland, B., Estepa, E.J., Emr, S.D., 1998. The Vps4p AAA
ATPase regulates membrane association of a Vps protein complex required
for normal endosome function. EMBO J. 17 (11), 29822993.
Babst, M., Katzmann, D.J., Estepa-Sabal, E.J., Meerloo, T., Emr, S.D., 2002a.
ESCRT-III: An endosome-associated heterooligomeric protein complex
required for MVB sorting. Dev. Cell 3 (2), 271282.
Babst, M., Katzmann, D.J., Snyder, W.B., Wendland, B., Emr, S.D., 2002b.
Endosome-associated complex, ESCRT-II, recruits transport machinery for
protein sorting at the multivesicular body. Dev. Cell 3 (2), 283289.
Bache, K.G., Brech, A., Mehlum, A., Stenmark, H., 2003. Hrs regulates
multivesicular body formation via ESCRT recruitment to endosomes.
J. Cell Biol. 162 (3), 435442.
Bamberg, S., Kolesnikova, L., Moller, P., Klenk, H.-D., Becker, S., 2005. VP24
of Marburg virus influences the formation of infectious particles. J. Virol.
79 (21).
Bavari, S., Bosio, C.M., Wiegand, E., Ruthel, G., Will, A.B., Geisbert, T.W.,
Hevey, M., Schmaljohn, C., Schmaljohn, A., Aman, M.J., 2002. Lipid raft
microdomains: a gateway for compartmentalized trafficking of Ebola and
Marburg viruses. J. Exp. Med. 195 (5), 593602.
Becker, S., Huppertz, S., Klenk, H.D., Feldmann, H., 1994. The nucleoprotein
of Marburg virus is phosphorylated. J. Gen. Virol. 75 (Pt. 4), 809818.
Becker, S., Rinne, C., Hofsass, U., Klenk, H.-D., Muhlberger, E., 1998.
Interactions of Marburg virus nucleocapsid proteins. Virology 249 (2),
406417.
Coronel, E.C., Takimoto, T., Murti, K.G., Varich, N., Portner, A., 2001.
Nucleocapsid incorporation into parainfluenza virus is regulated by specific
interaction with matrix protein. J. Virol. 75 (3), 11171123.
Demirov, D.G., Ono, A., Orenstein, J.M., Freed, E.O., 2002. Overexpression of
the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late
domain function. Proc. Natl. Acad. Sci. U.S.A. 99 (2), 955960.
Dessen, A., Volchkov, V., Dolnik, O., Klenk, H.D., Weissenhorn, W., 2000.
Crystal structure of the matrix protein VP40 from Ebola virus. EMBO J. 19
(16), 42284236.
Elliott, L.H., Kiley, M.P., McCormick, J.B., 1985. Descriptive analysis of Ebola
virus proteins. Virology 147 (1), 169176.
Feldmann, H., Kiley, M.P., 1999. Classification, structure, and replication of
filoviruses. Curr. Top. Microbiol. Immunol. 235, 121.
Feldmann, H., Will, C., Schikore, M., Slenczka, W., Klenk, H.D., 1991.
Glycosylation and oligomerization of the spike protein of Marburg virus.
Virology 182 (1), 353356.
Feldmann, H., Jones, S., Klenk, H.D., Schnittler, H.J., 2003. Ebola virus: from
discovery to vaccine. Nat. Rev., Immunol. 3 (8), 677685.
Feldmann, H., Wahl-Jensen, V., Jones, S.M., Stroher, U., 2004. Ebola virus
ecology: a continuing mystery. Trends Microbiol. 12 (10), 433437.
Funke, C., Becker, S., Dartsch, H., Klenk, H.D., Muhlberger, E., 1995.
Acylation of the Marburg virus glycoprotein. Virology 208 (1), 289297.
Garrus, J.E., von Schwedler, U.K., Pornillos, O.W., Morham, S.G., Zavitz,
irology 344 (2006) 6470K.H., Wang, H.E., Wettstein, D.A., Stray, K.M., Cote, M., Rich, R.L., 2001.
Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1
budding. Cell 107 (1), 5565.
/ VGeisbert, T.W., Jahrling, P.B., 1995. Differentiation of filoviruses by electron
microscopy. Virus Res. 39 (23), 129150.
Gomis-Ruth, F.X., Dessen, A., Timmins, J., Bracher, A., Kolesnikowa, L.,
Becker, S., Klenk, H.D., Weissenhorn, W., 2003. The matrix protein VP40
from Ebola virus octamerizes into pore-like structures with specific RNA
binding properties. Structure 11 (4), 423433.
Gottlinger, H.G., Dorfman, T., Sodroski, J.G., Haseltine, W.A., 1991. Effect of
mutations affecting the p6 gag protein on human immunodeficiency virus
particle release. Proc. Natl. Acad. Sci. U.S.A. 88 (8), 31953199.
Gruenberg, J., Stenmark, H., 2004. The biogenesis of multivesicular endo-
somes. Nat. Rev., Mol. Cell Biol. 5 (4), 317323.
Han, Z., Boshra, H., Sunyer, J.O., Zwiers, S.H., Paragas, J., Harty, R.N., 2003.
Biochemical and functional characterization of the Ebola virus VP24
protein: implications for a role in virus assembly and budding. J. Virol. 77
(3), 17931800.
Harty, R.N., Brown, M.E., Wang, G., Huibregtse, J., Hayes, F.P., 2000. A PPxY
motif within the VP40 protein of Ebola virus interacts physically and
functionally with a ubiquitin ligase: implications for filovirus budding.
Proc. Natl. Acad. Sci. U.S.A. 97 (25), 1387113876.
Hoenen, T., Volchkov, V., Kolesnikova, L., Mittler, E., Timmins, J., Ottmann,
M., Reynard, O., Becker, S., Weissenhorn, W., 2005. VP40 octamers are
essential for Ebola virus replication. J. Virol. 79 (3), 18981905.
Huang, M., Orenstein, J., Martin, M., Freed, E., 1995. p6Gag is required for
particle production from full-length human immunodeficiency virus type 1
molecular clones expressing protease. J. Virol. 69, 68106818.
Huang, Y., Xu, L., Sun, Y., Nabel, G.J., 2002. The assembly of Ebola virus
nucleocapsid requires virion-associated proteins 35 and 24 and posttrans-
lational modification of nucleoprotein. Mol. Cell 10 (2), 307316.
Ito, H., Watanabe, S., Takada, A., Kawaoka, Y., 2001. Ebola virus glycoprotein:
proteolytic processing, acylation, cell tropism, and detection of neutralizing
antibodies. J. Virol. 75 (3), 15761580.
Jasenosky, L.D., Neumann, G., Lukashevich, I., Kawaoka, Y., 2001. Ebola
virus VP40-induced particle formation and association with the lipid
bilayer. J. Virol. 75 (11), 52055214.
Kaptur, P.E., Rhodes, R.B., Lyles, D.S., 1991. Sequences of the vesicular
stomatitis virus matrix protein involved in binding to nucleocapsids.
J. Virol. 65 (3), 10571065.
Katzmann, D.J., Babst, M., Emr, S.D., 2001. Ubiquitin-dependent sorting into
the multivesicular body pathway requires the function of a conserved
endosomal protein sorting complex, ESCRT-I. Cell 106 (2), 145155.
Katzmann, D.J., Odorizzi, G., Emr, S.D., 2002. Receptor downregulation and
multivesicular-body sorting. Nat. Rev., Mol. Cell Biol. 3 (12), 893905.
Katzmann, D.J., Stefan, C.J., Babst, M., Emr, S.D., 2003. Vps27 recruits
ESCRT machinery to endosomes during MVB sorting. J. Cell Biol. 162 (3),
413423.
Katzmann, D.J., Sarkar, S., Chu, T., Audhya, A., Emr, S.D., 2004. Multi-
vesicular body sorting: ubiquitin ligase Rsp5 is required for the modifica-
tion and sorting of carboxypeptidase S. Mol. Biol. Cell 15 (2), 468480.
Kolesnikova, L., Becker, S., 2004. Virus maturation. In: Klenk, H.-D.,
Feldmann, H. (Eds.), Ebola and Marburg Viruses. Horizon Bioscience,
Norfolk, pp. 171203.
Kolesnikova, L., Muhlberger, E., Ryabchikova, E., Becker, S., 2000.
Ultrastructural organization of recombinant Marburg virus nucleoprotein:
comparison with Marburg virus inclusions. J. Virol. 74 (8), 38993904.
Kolesnikova, L., Bugany, H., Klenk, H.D., Becker, S., 2002. VP40, the matrix
protein of Marburg virus, is associated with membranes of the late
endosomal compartment. J. Virol. 76 (4), 18251838.
Kolesnikova, L., Bamberg, S., Berghofer, B., Becker, S., 2004a. The matrix
protein of Marburg virus is transported to the plasma membrane along
cellular membranes: exploiting the retrograde late endosomal pathway.
J. Virol. 78 (5), 23822393.
Kolesnikova, L., Berghofer, B., Bamberg, S., Becker, S., 2004b. Multivesicular
bodies as a platform for formation of the Marburg virus envelope. J. Virol.
78 (22), 1227712287.
Licata, J.M., Simpson-Holley, M., Wright, N.T., Han, Z., Paragas, J., Harty,
B. Hartlieb, W. WeissenhornR.N., 2003. Overlapping motifs (PTAP and PPEY) within the Ebola virus
VP40 protein function independently as late budding domains: involvement
of host proteins TSG101 and VPS-4. J. Virol. 77 (3), 18121819.Licata, J.M., Johnson, R.F., Han, Z., Harty, R.N., 2004. Contribution of Ebola
virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like
particles. J. Virol. 78 (14), 73447351.
Lin, Y., Kimpler, L.A., Naismith, T.V., Lauer, J.M., Hanson, P.I., 2005.
Interaction of the mammalian endosomal sorting complex required for
transport (ESCRT) III protein hSnf7-1 with itself, membranes, and the
AAA + ATPase SKD1. J. Biol. Chem. 280 (13), 1279912809.
Lu, Q., Hope, L.W., Brasch, M., Reinhard, C., Cohen, S.N., 2003. TSG101
interaction with HRS mediates endosomal trafficking and receptor down-
regulation. Proc. Natl. Acad. Sci. U.S.A. 100 (13), 76267631.
Martin-Serrano, J., Zang, T., Bieniasz, P., 2001. HIV-1 and Ebola virus encode
small peptide motifs that recruit Tsg101 to sites of particle assembly to
facilitate egress. Nat. Med. 7, 13131319.
Martin-Serrano, J., Yarovoy, A., Perez-Caballero, D., Bieniasz, P.D., 2003a.
Divergent retroviral late-budding domains recruit vacuolar protein sorting
factors by using alternative adaptor proteins. Proc. Natl. Acad. Sci. U.S.A.
100 (21), 1241412419.
Martin-Serrano, J., Zang, T., Bieniasz, P.D., 2003b. Role of ESCRT-I in
retroviral budding. J. Virol. 77 (8), 47944804.
Martin-Serrano, J., Perez-Caballero, D., Bieniasz, P.D., 2004. Context-
dependent effects of L domains and ubiquitination on viral budding.
J. Virol. 78 (11), 55545563.
Martin-Serrano, J., Eastman, S.W., Chung, W., Bieniasz, P.D., 2005. HECT
ubiquitin ligases link viral and cellular PPXY motifs to the vacuolar
protein-sorting pathway. J. Cell Biol. 168 (1), 89101.
Mavrakis, M., Kolesnikova, L., Schoehn, G., Becker, S., Ruigrok, R.W., 2002.
Morphology of Marburg virus NPRNA. Virology 296 (2), 300307.
Modrof, J., Muhlberger, E., Klenk, H.D., Becker, S., 2002. Phosphorylation
of VP30 impairs Ebola virus transcription. J. Biol. Chem. 277 (36),
3309933104.
Morita, E., Sundquist, W.I., 2004. Retrovirus budding. Ann. Rev. Cell Dev.
Biol. 20 (1), 395425.
Neumann, G., Ebihara, H., Takada, A., Noda, T., Kobasa, D., Jasenosky, L.D.,
Watanabe, S., Kim, J.H., Feldmann, H., Kawaoka, Y., 2005. Ebola virus
VP40 late domains are not essential for viral replication in cell culture.
J. Virol. 79 (16), 1030010307.
Nguyen, T.L., Schoehn, G., Weissenhorn, W., Hermone, A.R., Burnett, J.C.,
Panchal, R.G., McGrath, C., Zaharevitz, D.W., Aman, M.J., Gussio, R.,
Bavari, S., 2005. An all-atom model of the pore-like structure of hexameric
VP40 from Ebola: structural insights into the monomerhexamer transi-
tion. J. Struct. Biol. 151, 3040.
Noda, T., Sagara, H., Suzuki, E., Takada, A., Kida, H., Kawaoka, Y., 2002.
Ebola virus VP40 drives the formation of virus-like filamentous particles
along with GP. J. Virol. 76 (10), 48554865.
Noda, T., Aoyama, K., Sagara, H., Kida, H., Kawaoka, Y., 2005. Nucleocapsid-
like structures of Ebola virus reconstructed using electron tomography.
J. Vet. Med. Sci. 67 (3), 325328.
Ott, D.E., Coren, L.V., Copeland, T.D., Kane, B.P., Johnson, D.G., Sowder II,
R.C., Yoshinaka, Y., Oroszlan, S., Arthur, L.O., Henderson, L.E., 1998.
Ubiquitin is covalently attached to the p6Gag proteins of human
immunodeficiency virus type 1 and simian immunodeficiency virus and
to the p12Gag protein of Moloney murine leukemia virus. J. Virol. 72 (4),
29622968.
Ott, D.E., Coren, L.V., Chertova, E.N., Gagliardi, T.D., Schubert, U.,
2000. Ubiquitination of HIV-1 and MuLV Gag. Virology 278 (1),
111121.
Panchal, R.G., Ruthel, G., Kenny, T.A., Kallstrom, G.H., Lane, D., Badie, S.S.,
Li, L., Bavari, S., Aman, M.J., 2003. In vivo oligomerization and raft
localization of Ebola virus protein VP40 during vesicular budding. Proc.
Natl. Acad. Sci. U.S.A. 100 (26), 1593615941.
Parent, L., Bennett, R., Craven, R., Nelle, T., Krishna, N., Bowzard, J., Wilson,
C., Puffer, B., Montelaro, R., Wills, J., 1995. Positionally independent and
exchangeable late budding functions of the Rous sarcoma virus and human
immunodeficiency virus Gag proteins. J. Virol. 69, 54555460.
Patnaik, A., Chau, V., Wills, J., 2000. Ubiquitin is part of the retrovirus budding
irology 344 (2006) 6470 69machinery. Proc. Natl. Acad. Sci. U.S.A. 97, 1306913074.
Putterman, D., Pepinsky, R.B., Vogt, V.M., 1990. Ubiquitin in avian leukosis
virus particles. Virology 176 (2), 633637.
Ruigrok, R.W.H., Schoehn, G., Dessen, A., Forest, E., Volchkov, V., Dolnik,
O., Klenk, H.D., Weissenhorn, W., 2000. Structural characterization and
membrane binding properties of the matrix protein VP40 of Ebola virus.
J. Mol. Biol. 300 (1), 103112.
Ruthel, G., Demmin, G.L., Kallstrom, G., Javid, M.P., Badie, S.S., Will, A.B.,
Nelle, T., Schokman, R., Nguyen, T.L., Carra, J.H., Bavari, S., Aman, M.J.,
2005. Association of Ebola virus matrix protein VP40 with microtubules.
J. Virol. 79 (8), 47094719.
Sanchez, A., Kiley, M.P., Klenk, H.D., Feldmann, H., 1992. Sequence
analysis of the Marburg virus nucleoprotein gene: comparison to Ebola
virus and other non-segmented negative-strand RNA viruses. J. Gen.
Virol. 73, 347357.
Sanger, C., Muhlberger, E., Ryabchikova, E., Kolesnikova, L., Klenk, H.D.,
Becker, S., 2001. Sorting of Marburg virus surface protein and virus release
take place at opposite surfaces of infected polarized epithelial cells. J. Virol.
Swenson, D.L., Warfield, K.L., Kuehl, K., Larsen, T., Hevey, M.C.,
Schmaljohn, A., Bavari, S., Aman, M.J., 2004. Generation of Marburg
virus-like particles by co-expression of glycoprotein and matrix protein.
FEMS Immunol. Med. Microbiol. 40 (1), 2731.
Tanzi, G.O., Piefer, A.J., Bates, P., 2003. Equine infectious anemia virus
utilizes host vesicular protein sorting machinery during particle release.
J. Virol. 77 (15), 84408447.
Timmins, J., Scianimanico, S., Schoehn, G., Weissenhorn, W., 2001. Vesicular
release of Ebola virus matrix protein VP40. Virology 283 (1), 16.
Timmins, J., Schoehn, G., Kohlhaas, C., Klenk, H.D., Ruigrok, R.W.H.,
Weissenhorn, W., 2003a. Oligomerization and polymerization of the
filovirus matrix protein VP40. Virology 312 (2), 359368.
Timmins, J., Schoehn, G., Ricard-Blum, S., Scianimanico, S., Vernet, T.,
Ruigrok, R.W.H., Weissenhorn, W., 2003b. Ebola virus matrix protein
VP40 interaction with human cellular factors Tsg101 and Nedd4. J. Mol.
Biol. 326 (2), 493502.
B. Hartlieb, W. Weissenhorn / Virology 344 (2006) 647070Schibli, D.J., Weissenhorn, W., 2004. Class I and class II viral fusion protein
structures reveal similar principles in membrane fusion. Mol. Membr. Biol.
21 (6), 361371.
Schmitt, A.P., Lamb, R.A., 2004. Escaping from the cell: assembly and
budding of negative-strand RNA viruses. Curr. Top. Microbiol. Immunol.
283, 145196.
Schmitt, A.P., Leser, G.P., Waning, D.L., Lamb, R.A., 2002. Requirements for
budding of paramyxovirus simian virus 5 virus-like particles. J. Virol. 76
(8), 39523964.
Schubert, U., Ott, D.E., Chertova, E.N., Welker, R., Tessmer, U., Princiotta,
M.F., Bennink, J.R., Krausslich, H.G., Yewdell, J.W., 2000. Proteasome
inhibition interferes with gag polyprotein processing, release, and
maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. U.S.A. 97 (24),
1305713062.
Scianimanico, S., Schoehn, G., Timmins, J., Ruigrok, R.H., Klenk, H.D.,
Weissenhorn, W., 2000. Membrane association induces a conformational
change in the Ebola virus matrix protein. EMBO J. 19 (24), 67326741.
Strack, B., Calistri, A., Accola, M., Palu, G., Gottlinger, H., 2000. A role for
ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci.
U.S.A. 97, 1306313068.
Strack, B., Calistri, A., Craig, S., Popova, E., Gottlinger, H.G., 2003.
AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in
virus budding. Cell 114 (6), 689699.
Stricker, R., Mottet, G., Roux, L., 1994. The Sendai virus matrix protein
appears to be recruited in the cytoplasm by the viral nucleocapsid to
function in viral assembly and budding. J. Gen. Virol. 75, 10311042.
Stuchell, M.D., Garrus, J.E., Muller, B., Stray, K.M., Ghaffarian, S.,
McKinnon, R., Krausslich, H.G., Morham, S.G., Sundquist, W.I., 2004.
The human endosomal sorting complex required for transport (ESCRT-I)
and its role in HIV-1 budding. J. Biol. Chem. 279 (34), 3605936071.
Sundquist, W.I., Schubert, H.L., Kelly, B.N., Hill, G.C., Holton, J.M., Hill,
C.P., 2004. Ubiquitin recognition by the human TSG101 protein. Mol. Cell
13 (6), 783789.VerPlank, L., Bouamr, F., LaGrassa, T.J., Agresta, B., Kikonyogo, A., Leis, J.,
Carter, C.A., 2001. Tsg101, a homologue of ubiquitin-conjugating (E2)
enzymes, binds the L domain in HIV type 1 Pr55Gag. Proc. Natl. Acad. Sci.
U.S.A. 98 (14), 77247729.
Volchkov, V.E., Feldmann, H., Volchkova, V.A., Klenk, H.D., 1998a.
Processing of the Ebola virus glycoprotein by the proprotein convertase
furin. Proc. Natl. Acad. Sci. U.S.A. 95 (10), 57625767.
Volchkov, V.E., Volchkova, V.A., Slenczka, W., Klenk, H.D., Feldmann, H.,
1998b. Release of viral glycoproteins during Ebola virus infection.
Virology 245 (1), 110119.
Volchkov, V.E., Volchkova, V.A., Stroher, U., Becker, S., Dolnik, O., Cieplik,
M., Garten, W., Klenk, H.D., Feldmann, H., 2000. Proteolytic processing of
Marburg virus glycoprotein. Virology 268 (1), 16.
von Schwedler, U.K., Stuchell, M., Muller, B., Ward, D.M., Chung, H.Y.,
Morita, E., Wang, H.E., Davis, T., He, G.P., Cimbora, D.M., Scott, A.,
Krausslich, H.G., Kaplan, J., Morham, S.G., Sundquist, W.I., 2003. The
protein network of HIV budding. Cell 114 (6), 701713.
Warfield, K.L., Bosio, C.M., Welcher, B.C., Deal, E.M., Mohamadzadeh, M.,
Schmaljohn, A., Aman, M.J., Bavari, S., 2003. Ebola virus-like particles
protect from lethal Ebola virus infection. Proc. Natl. Acad. Sci. U.S.A. 100
(26), 1588915894.
Warfield, K.L., Swenson, D.L., Negley, D.L., Schmaljohn, A.L., Aman, M.J.,
Bavari, S., 2004. Marburg virus-like particles protect guinea pigs from
lethal Marburg virus infection. Vaccine 22 (2526), 34953502.
Watanabe, S., Watanabe, T., Noda, T., Takada, A., Feldmann, H., Jasenosky,
L.D., Kawaoka, Y., 2004. Production of novel Ebola virus-like particles
from cDNAs: an alternative to Ebola virus generation by reverse genetics.
J. Virol. 78 (2), 9991005.
Wills, J., Cameron, C., Wilson, C., Xiang, Y., Bennett, R., Leis, J., 1994. An
assembly domain of the Rous sarcoma virus Gag protein required late in
budding. J. Virol. 68, 66056618.
Yasuda, J., Nakao, M., Kawaoka, Y., Shida, H., 2003. Nedd4 regulates egress
of Ebola virus-like particles from host cells. J. Virol. 77 (18), 99879992.75 (3), 12741283.
Filovirus assembly and buddingIntroductionDifferent conformations of the matrix protein VP40Cellular factors implicated in enveloped virus buddingVP40 interaction with cellular factorsCellular transport of VP40The role of the glycoprotein GP in assembly and buddingAssembly of the nucleocapsidConclusionsAcknowledgmentsReferences