Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells.
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.
Charpilienne
A
A
Virologie Moléculaire et Cellullaire, INRA, 78352 Jouy-en-Josas, Cedex, France.
Nejmeddine
M
M
Berois
M
M
Parez
N
N
Neumann
E
E
Hewat
E
E
Trugnan
G
G
Cohen
J
J
D000818:Animals; D000956:Antigens, Viral; D016367:Baculoviridae / Q000502:physiology / Q000648:ultrastructure; D002213:Capsid / Q000032:analysis* / Q000235:genetics; D036022:Capsid Proteins; D002460:Cell Line; D020285:Cryoelectron Microscopy; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012401:Rotavirus / Q000648:ultrastructure*; D018411:Spodoptera; D014162:Transfection
D000956:Antigens, Viral; D036022:Capsid Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C442545:VP2 protein, Rotavirus; C060339:VP4 protein, Rotavirus; C061332:VP6 protein, Rotavirus; C050779:VP7 protein, Rotavirus; D049452:Green Fluorescent Proteins