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: weissen@embl-grenoble.fr (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.

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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