3FNC1†Present address: Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Research Laboratories, 40 Smith Walk, Philadelphia, PA 19104, USA.
The matrix (M) protein of vesicular stomatitis virus plays a key role in both assembly and budding of progeny virions. In vitro experiments have shown a strong propensity of M protein to bind to vesicles containing negatively charged phospholipids. In vivo, it has also been demonstrated that recruitment of some cellular proteins by M protein is required for efficient virus budding and release of newly synthesized virions in the extracellular medium. The ability of M protein to deform target membranes in vitro was investigated in this study. It was shown that incubation of purified M protein with giant unilamellar vesicles results in the formation of patches of M protein at their surface, followed by deformations of the membrane toward the inside of the vesicle, which could be observed in phase-contrast microscopy. This provides the first evidence that M protein alone is able to impose the correct budding curvature on the membrane. Using confocal microscopy, patches of M protein that colocalized with negatively charged lipid domains a few minutes after vesicle injection were observed. After a longer incubation period, membrane deformations appeared in these domains. At this time, a strict colocalization of M protein, negatively charged lipids and membrane deformation was observed. The influence on this process of the basic N-terminal part of the protein and of the previously identified hydrophobic loop has also been investigated. Interestingly, the final fission event has never been observed in our experimental system, indicating that other partners are required for this step.
BaudinF.,
PetitI.,
WeissenhornW.,
RuigrokR. W.2001; In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology 281:102–108[CrossRef]
BirdwellC. R.,
StraussJ. H.1974; Maturation of vesicular stomatitis virus: electron microscopy of surface replicas of infected cells. Virology 59:587–590[CrossRef]
BlotV.,
PerugiF.,
GayB.7 other authors2004; Nedd4.1-mediated ubiquitination and subsequent recruitment of Tsg101 ensure HTLV-1 Gag trafficking towards the multivesicular body pathway prior to virus budding. J Cell Sci 117:2357–2367[CrossRef]
BuechiM.,
BachiT.1982; Microscopy of internal structures of Sendai virus associated with the cytoplasmic surface of host membranes. Virology 120:349–359[CrossRef]
CravenR. C.,
HartyR. N.,
ParagasJ.,
PaleseP.,
WillsJ. W.1999; Late domain function identified in the vesicular stomatitis virus M protein by use of rhabdovirus-retrovirus chimeras. J Virol 73:3359–3365
DenisovG.,
WanaskiS.,
LuanP.,
GlaserM.,
McLaughlinS.1998; Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: an electrostatic model and experimental results. Biophys J 74:731–744[CrossRef]
DessenA.,
VolchkovV.,
DolnikO.,
KlenkH. D.,
WeissenhornW.2000; Crystal structure of the matrix protein VP40 from Ebola virus. EMBO J 19:4228–4236[CrossRef]
GarrusJ. E.,
von SchwedlerU. K.,
PornillosO. W.9 other authors2001; Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107:55–65[CrossRef]
GaudinY.,
BargeA.,
EbelC.,
RuigrokR. W.1995; Aggregation of VSV M protein is reversible and mediated by nucleation sites: implications for viral assembly. Virology 206:28–37[CrossRef]
Gomez-PuertasP.,
AlboC.,
Perez-PastranaE.,
VivoA.,
PortelaA.2000; Influenza virus matrix protein is the major driving force in virus budding. J Virol 74:11538–11547[CrossRef]
HartyR. N.,
ParagasJ.,
SudolM.,
PaleseP.1999; A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J Virol 73:2921–2929
HeggenessM. H.,
SmithP. R.,
ChoppinP. W.1982; In vitro assembly of the nonglycosylated membrane protein (M) of Sendai virus. Proc Natl Acad Sci U S A 79:6232–6236[CrossRef]
JayakarH. R.,
MurtiK. G.,
WhittM. A.2000; Mutations in the PPPY motif of vesicular stomatitis virus matrix protein reduce virus budding by inhibiting a late step in virion release. J Virol 74:9818–9827[CrossRef]
LenardJ.,
VanderoefR.1990; Localization of the membrane-associated region of vesicular stomatitis virus M protein at the N terminus, using the hydrophobic, photoreactive probe 125I-TID. J Virol 64:3486–3491
LuanP.,
YangL.,
GlaserM.1995; Formation of membrane domains created during the budding of vesicular stomatitis virus. A model for selective lipid and protein sorting in biological membranes. Biochemistry 34:9874–9883[CrossRef]
MathivetL.,
CribierS.,
DevauxP. F.1996; Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys J 70:1112–1121[CrossRef]
MebatsionT.,
WeilandF.,
ConzelmannK. K.1999; Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G. J Virol 73:242–250
RouxA.,
CuvelierD.,
NassoyP.,
ProstJ.,
BassereauP.,
GoudB.2005; Role of curvature and phase transition in lipid sorting and fission of membrane tubules. EMBO J 24:1537–1545[CrossRef]
ScianimanicoS.,
SchoehnG.,
TimminsJ.,
RuigrokR. H.,
KlenkH. D.,
WeissenhornW.2000; Membrane association induces a conformational change in the Ebola virus matrix protein. EMBO J 19:6732–6741[CrossRef]
TimminsJ.,
SchoehnG.,
KohlhaasC.,
KlenkH. D.,
RuigrokR. W.,
WeissenhornW.2003a; Oligomerization and polymerization of the filovirus matrix protein VP40. Virology 312:359–368[CrossRef]
TimminsJ.,
SchoehnG.,
Ricard-BlumS.,
ScianimanicoS.,
VernetT.,
RuigrokR. W.,
WeissenhornW.2003b; Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4. J Mol Biol 326:493–502[CrossRef]
ZakowskiJ. J.,
PetriW. A.Jr,
WagnerR. R.1981; Role of matrix protein in assembling the membrane of vesicular stomatitis virus: reconstitution of matrix protein with negatively charged phospholipid vesicles. Biochemistry 20:3902–3907[CrossRef]
ZhangJ.,
LambR. A.1996; Characterization of the membrane association of the influenza virus matrix protein in living cells. Virology 225:255–266[CrossRef]