Individual Rotavirus-like Particles Containing 120 Molecules of Fluorescent Protein Are Visible in Living Cells

Annie Charpilienne,Mohamed Nejmeddine,Mabel Berois,Nathalie Parez,Emmanuelle Neumann,Elizabeth Hewat,Germain Trugnan,Jean Cohen

doi:10.1074/jbc.m101935200

Abstract

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.

Highlights

The rotaviruses, members of the family Reoviridae, are the most important cause of severe viral gastroenteritis in humans and animals
Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP-VP2 auto assembles perfectly well and forms fluorescent virus-like particles (VLP) (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6
Three chimeric VLP have been constructed with exceptionally large inserts; they are papillomavirus VLP containing E7 and E2 of the same virus [14], parvovirus B19 with hen egg white lysozyme presented at the capsid surface [15], and hepatitis B VLP with green fluorescent protein (GFP) inserted on an external loop [16]

Summary

Fluorescent Rotavirus VLP

We prepared two chimeric VP2 in which amino acids 1–92 are replaced either by the entire 238-aa GFP or by the entire 249-aa DsRed protein [21], both followed by a flexible linker. This addition did not affect assembly of double and triple layered VLP. A single VLP can be detected by fluorescent microscopy. This is the first report of VLP with a long exogenous polypeptide enclosed within. These chimeric particles allow to study the first steps of rotavirus infection

EXPERIMENTAL PROCEDURES

To generate the recombinant baculovirus expressing GFP fused to

RESULTS

DISCUSSION