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.

Two types of particles were isolated during purification of rotavirus. Dense (D) particles have a density of 1.38 in CsCl and exhibit spontaneously a fully active endogenous transcriptase. Light (L) particles (density of 1.36 in CsCl) need to be treated with chelating agents to show a polymerase activity. The activation process of L particles was studied under strictly controlled monovalent, divalent, and hydrogen ion concentrations. These experiments demonstrate that i) activation is not affected by the ionic strength ii) activation occurs only at a pH higher than 7.1 iii) a low concentration of chelating agent (40 muM EDTA) is sufficient to activate the enzyme. Treatment of particles with EGTA, which chelates selectively Ca2+, leads to unmasking even in the presence of magnesium, indicating that the concentration of free calcium ions plays a major role in the activation process. Various glycosidases, detergents, and chelating agents were tested in respect to unmasking properties. Of these compound only chelating agents turned out to be efficient. Following activation, two glycopeptides were solubilized. These glycopeptides have an apparent molecular weight of 34,000 and 31,000 daltons and react with concanavalin A. The role of Ca2+ upon the stability of virus particles, and the activation of the endogenous transcriptase in vitro and in the infected cells is discussed.

Human rotavirus-specific CD4؉ and CD8 ؉ T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8؉ and CD4 ؉ T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4 ؉ and CD8 ؉ T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4؉ and CD8 ؉ T cells and the frequencies of IL-13-secreting CD4 ؉ T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4 ؉ cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.Rotaviruses (RV) are the most important cause of severe dehydrating diarrhea in children worldwide, resulting in an estimated 480,000 to 640,000 deaths annually (5). The first vaccine approved for use in humans has recently been withdrawn by the manufacturer because it was associated with increased numbers of cases of intussusception (1). For these reasons, investigators are exploring alternative vaccination strategies to develop improved second-generation vaccine candidates. A detailed knowledge of the immune response against RV in humans will be useful for the design and/or evaluation of new RV vaccines.The RV-specific cell-mediated immune response is relatively well studied in the murine model: CD4 ϩ T cells are essential for the development of more than 90% of the RV-specific intestinal immunoglobulin A (IgA) (14). Moreover, the antibody response seems to be the main mechanism that mediates protection against RV reinfection (14,15,31). Murine RVspecific CD8 ϩ T cells have a direct antiviral effect, are involved in the timely resolution of primary RV infection, and can mediate partial protection against reinfection (13,15,31).Because RV replication is restricted to...

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03–0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with Kd < 1 × 10−10). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.

Among Caliciviridae, the norovirus genus encompasses enteric viruses that infect humans as well as several animal species, causing gastroenteritis. Porcine strains are classified together with human strains within genogroup II, whilst bovine norovirus strains represent genogroup III. Various GI and GII human strains bind to carbohydrates of the histo-blood group family which may be shared among mammalian species. Genetic relatedness of human and animal strains as well as the presence of potentially shared ligands raises the possibility of norovirus cross-species transmission. In the present study, we identified a carbohydrate ligand for the prototype bovine norovirus strain Bo/Newbury2/76/UK (NB2). Attachment of virus-like particles (VLPs) of the NB2 strain to bovine gut tissue sections showed a complete match with the staining by reagents recognizing the Galα1,3 motif. Alpha-galactosidase treatment confirmed involvement of a terminal alpha-linked galactose. Specific binding of VLPs to the αGal epitope (Galα3Galβ4GlcNAcβ-R) was observed. The binding of Galα3GalαOMe to rNB2 VLPs was characterized at atomic resolution employing saturation transfer difference (STD) NMR experiments. Transfection of human cells with an α1,3galactosyltransferase cDNA allowed binding of NB2 VLPs, whilst inversely, attachment to porcine vascular endothelial cells was lost when the cells originated from an α1,3galactosyltransferase KO animal. The αGal epitope is expressed in all mammalian species with the exception of the Hominidaea family due to the inactivation of the α1,3galactosyltransferase gene (GGTA1). Accordingly, the NB2 carbohydrate ligand is absent from human tissues. Although expressed on porcine vascular endothelial cells, we observed that unlike in cows, it is not present on gut epithelial cells, suggesting that neither man nor pig could be infected by the NB2 bovine strain.

The complete VP2 gene of bovine rotavirus strain RF has been inserted into the baculovirus transfer vector pVL941 under the control of the polyhedrin promoter. Cotransfection of Spodoptera frugiperda 9 cells with wild-type baculovirus DNA and transfer vector DNA led to the formation of recombinant baculoviruses which contain bovine rotavirus gene 2. Infection of S. frugiperda cells with this recombinant virus resulted in the production of a protein similar in size and antigenic properties to the authentic rotavirus VP2. The protein binds double-stranded RNA and DNA in an overlay protein blot assay. Expressed VP2 assembles in the cytoplasm of infected cells in corelike particles 45 nm in diameter. These corelike particles were purified by sucrose gradient centrifugation and found to be devoid of nucleic acid. Coexpression of VP2 and VP6 from heterologous rotavirus strains (bovine and simian) resulted in the formation of single-shelled particles. These results definitively show the existence of an innermost protein shell in rotavirus which is formed independently of other rotavirus proteins. These results have implications for schemes of rotavirus morphogenesis.

Rotavirus has a complex triple-layered icosahedral capsid. The external layer consists of VP7 and VP4, the intermediate layer consists of VP6 trimers, and the internal layer consists of VP2. Double-layered particles (DLP) derived from the virus by solubilization of VP4 and VP7 are transcriptionally competent and extrude capped mRNA from their vertices. Analysis of the pseudoatomic model of the VP6 layer, obtained by placing the atomic structure of VP6 into electron microscopy reconstructions of the DLP, has identified the regions of the protein involved in interactions with the internal layer. To study the role of VP6 both in the assembly of DLP and in transcription, 13 site-specific substitution mutations of VP6, targeting the contacts between the two inner layers, were constructed and expressed in the baculovirus system. The effects of these mutations on VP6 expression, trimerization, and formation of macromolecular assemblies were investigated. Using either in vitro reconstituted DLP derived from purified viral cores and recombinant VP6 or in vivo self-assembled virus-like particles resulting from the coexpression of VP2 and VP6 in the baculovirus-Sf9 system (VLP2/6), we have identified the amino acids essential for recovery of transcription or assembly. All VP6 mutants formed stable trimers which, like wild-type VP6, assembled into tubular structures. The ability of VP6 to interact with VP2 was examined by several assays, including electron microscopy, coimmunoprecipitation, purification of VLP2/6, and monitoring of the transcriptase activity of reconstituted DLP. Of the 13 VP6 mutants examined, 3 were unable to assemble with VP2 and 3 others partially assembled. These mutants either did not rescue the transcriptase activity of core particles or did so only marginally. Four mutants as well as the wild-type VP6 assembled and transcribed very well. Three mutants assembled well on cores but, surprisingly, did not rescue the transcriptase activity of reconstituted DLP. Our results indicate that hydrophobic interactions between VP6 and VP2 residues are responsible for the stability of the DLP. They also show that subtle electrostatic interactions between VP6 and the underlying transcriptase machinery can be essential for mRNA synthesis.Rotaviruses are members of the Reoviridae family of segmented, double-stranded RNA viruses. Viruses in this family are nonenveloped, and their complex capsids contain several concentric protein layers displaying icosahedral symmetry. Structural studies by X-ray crystallography have revealed the complex interactions between the innermost layers of some of the Reoviridae, like bluetongue virus (BTV) (9), as well as the structure of the single-layered core particle of reoviruses (20). In the case of rotaviruses, the crystal structure of the doublelayered particle (DLP) has not yet been reported but a pseudoatomic model has been derived for the middle layer by use of an approach combining X-ray crystallography and electron microscopy (18). As is the case for BTV, the two innermost lay...

The self-assembly kinetics for a norovirus capsid protein were probed by time-resolved small-angle X-ray scattering and then analyzed by singular value decomposition and global fitting. Only three species contribute to the total scattering intensities: dimers, intermediates comprising some 11 dimers, and icosahedral T = 3 capsids made up of 90 dimers. Three-dimensional reconstructions of the intermediate robustly show a stave-like shape consistent with an arrangement of two pentameric units connected by an interstitial dimer. Upon triggering of self-assembly, the biphasic kinetics consist of a fast step in which dimers are assembled into intermediates, followed by a slow step in which intermediates interlock into capsids. This simple kinetic model reproduces experimental data with an excellent agreement over 6 decades in time and with nanometer resolution. The extracted form factors are robust against changes in experimental conditions. These findings challenge and complement currently accepted models for the assembly of norovirus capsids.