Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.Human adenoviruses (Ads) are nonenveloped viruses responsible for respiratory, ocular, and enteric infections. Their icosahedral capsid, containing the 36-kpb double-stranded DNA genome, is composed of three major proteins: the hexon, the penton (Pt) base, and the fiber. At the 12 vertices of the capsid, the protruding fiber is noncovalently attached to the Pt base, thus forming the Pt complex. It has been reported that the fiber interacts with a high affinity with a primary receptor and that the subsequent interaction of the Pt base RGD motif with cellular integrins triggers endocytosis (17). A 42-kDa protein, called the coxsackievirus and Ad receptor (CAR), is recognized by at least one serotype of each of the six subgroups of Ad except for the serotypes belonging to subgroup B (i.e., Ad3) (1, 12). Even though that Ad capsid is composed of at least 11 proteins, it has been shown that Pt alone can penetrate into human cell lines, thus making of this complex a potential vector for DNA delivery (2,5,7,8,16). Remarkably, Ad3 but not Ad5 Pt expressed in the baculovirus system led to the formation of symmetric complexes of 12 Pts called dodecahedron (Dd). We have previously shown that the non-CARbinding Ad3-Dd can be used as a gene transfer vector (2), and we show here that this virus-like particle is also able to transduce large multimeric proteins into human cells.Dd internalization. Coexpression of Ad3 Pt base and fiber proteins in the baculovirus system led to the formation of a symmetric dodecameric particle (2, 14), Pt-Dd (Fig. 1a). This complex results from the interaction between the pentameric base proteins, as attested by the Dd formation upon expression of base proteins alone (Bs-Dd; Fig. 1a). The respective roles of the fiber and the Pt base protein in Ad3 Dd entry into HeLa cells was assessed by immunofluorescence. HeLa cells grown in a 96-multiwell plate (Falcon) at 2 ϫ 10 4 cells per well were incubated in 100 l of phosphate-buffered saline (PBS) with a range of Dd concentrations. After 1 h at 37°C, cells were * Corresponding author. Mailing address: Institut de Biologie Structurale, 41 Rue Jules Horowitz, 38027 Grenoble,. E-mail: fender@ibs.fr.
A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixationpermeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several malignancies (including Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas) in transplant recipients and patients infected with human immunodeficiency virus (28). EBV infects normal resting B cells in vitro and activates them in a way reminiscent of that in which B cells are activated in response to their cognate antigen. The resulting immortalized lymphoblastoid cell lines (LCLs) express a well-defined phenotype characterized by the expression of high levels of activation and adhesion molecules and resemble lymphoblasts (22). The virus achieves this by encoding six EBNAs, three latent membrane proteins (LMPs), and two untranslated RNAs (EBERs), not all of which are essential for immortalization. EBV remains latent in resting circulating B cells with its genome present in a limited number of copies as covalently closed episomes. The switch between latency and replication of EBV is mediated by the BZLF1 gene in the BamHIZ fragment of EBV. The BZLF1-encoded protein (ZEBRA) is a 38-kDa nuclear protein. Like ZEBRA (or Zta), the R transactivator (Rta) plays an important role in the switch from latency to the lytic phase (16). In both epithelial cells and B lymphocytes (21), Rta alone is capable of disrupting latency. In other cell types, a combination of ZEBRA and Rta induces the activation of early viral promoters (2). In studies based on serological data, we have demonstrated that high titers of anti-ZEBRA antibodies are to be found both in patients with Hodgkin's disease (6, 11) and before posttransplant lymphoproliferative disorders in renal transplant recipients (5).The presence of these two immediate-early proteins in the B-cell lineage, as detected with monoclonal antibodies produced in our laboratory (3), could thus be evaluated as a marker of virus replication, possibly associated with tumor progression (13). Infected cells in the peripheral blood of persistently infected healthy individuals are confined to the B-cell lineage and are present at levels varying between 1 and 50 per 10 6 B cells (18). In immunocompromised patients, they are present at higher levels, e.g., 1 to...
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