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...
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Published in | The Journal of biological chemistry Vol. 276; no. 31; pp. 29361 - 29367 |
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Main Authors | , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
03.08.2001
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Subjects | |
Online Access | Get full text |
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Summary: | 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 ânanoboxesâ carrying macromolecules
to living cells. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M101935200 |