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J. Biol. Chem., Vol. 276, Issue 31, 29361-29367, August 3, 2001
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From Rotaviruses are large, complex icosahedral
particles consisting of three concentric capsid layers. When the
innermost capsid protein VP2 is expressed in the baculovirus-insect
cell system it assembles as core-like particles. The amino terminus
region of VP2 is dispensable for assembly of virus-like particles
(VLP). Coexpression of VP2 and VP6 produces double layered VLP.
We hypothesized that the amino end of VP2 could be extended without
altering the auto assembly properties of VP2. Using the green
fluorescent protein (GFP) or the DsRed protein as model inserts we have
shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles
perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6)
when coexpressed with VP6. The presence of GFP inside the core does not
prevent the assembly of the outer capsid layer proteins VP7 and VP4 to
give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed
that GFP molecules are located at the 5-fold vertices of the core. It
is possible to visualize a single fluorescent VLP in living cells by
confocal fluorescent microscopy. In vitro VLP2/6 did not
enter into permissive cells or in dendritic cells. In contrast,
fluorescent VLP2/6/7/4 entered the cells and then the fluorescence
signal disappear rapidly. Presented data indicate that fluorescent VLP
are interesting tools to follow in real time the entry process of
rotavirus and that chimeric VLP could be envisaged as "nanoboxes"
carrying macromolecules to living cells.
Individual Rotavirus-like Particles Containing 120 Molecules of Fluorescent Protein Are Visible in Living
Cells*,
,§,,
,,**
Virologie Moléculaire et Cellullaire,
INRA, 78352 Jouy-en-Josas, Cedex, France, § INSERM
U538, Faculté de Médecine Saint-Antoine, 27 rue de
Chaligny, 75571 Paris, Cedex 12, France, ¶ Laboratoire de
Virologie, Hôpital Armand Trousseau (EA 2391, UFR Saint-Antoine),
Paris, France, and Laboratoire de Microscopie Electronique
Structurale, Institut de Biologie Structurale, 41 rue Jules Horowitz,
38027 Grenoble, Cedex 1, France
*
This work was supported by a Programme de Reherche
Fondamentale en Microbiologie, sur les Maladies Infectieuses et
Parasitaires grant (148-2000) from Ministere de l'EN seignement de la
Recherche et de la Technologie, a grant from Action Concertée
Initiative Microbiologie (1A029F), an Innovation Technique et
Methodologique grant (4TM06F) from INSERM, and a 5th Programme Cadre de
Recherche et Developpement grant from Union Européenne (QLRT
1999-00634).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a movie corresponding to
the whole sequence of GFP-VLP entry in dendritic cell (Fig. 9).
**
To whom correspondence should be addressed. Tel.: 33-0-1-3465-2604;
Fax: 33-0-1-3465-2621; E-mail: cohen@jouy.inra.fr.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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