Adenovirus Dodecahedron Allows Large Multimeric Protein Transduction in Human Cells

Abstract

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.

Structure of the Dodecahedral Penton Particle from Human Adenovirus Type 3

“…The fiber itself, however, was poorly resolved, possibly as a consequence of intrinsic flexibility, limited number of particles included in the reconstruction (30) or from bending imposed by a restricted depth of ice formed during preparation for microscopy. Subsequently, the dodecahedron was shown to enter cells efficiently using the same mechanism as adenovirus, 19,20 confirming the localization of the infection mechanisms to the penton's two constituent proteins, penton base and fiber. We have previously proposed the dodecahedron as an alternative to adenovirus for therapeutic delivery.…”

“…We have previously proposed the dodecahedron as an alternative to adenovirus for therapeutic delivery. 19,20 One of the potential advantages over the intact virus particle is that it cannot provoke infection directly or through recombination, as it harbors none of the viral genome. A second is the possibility of a weaker immune response compared to adenovirus because of its reduced protein content, thereby enhancing the success of multiple applications.…”

Heparan Sulphate Proteoglycans and Viral Vectors : Ally or Foe?

“…A clear advantage of this sub-viral particle is that it retains adenovirus endocytotic features but is devoid of genetic material. The proof of concept of Dd use for protein delivery has been made for large multimeric proteins, such as a monoclonal antibody [Fender et al, 2003] . Recently, a universal adaptor has been developed to make of Dd a versatile vector for protein transduction [Garcel et al, in press].…”

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

“…3 ). While adenovirus dodecahedron's biological function is still not fully understood, this versatile virus-like particle can be used for biotechnology applications [17][18][19][20] or as a tool for receptor identification [8] . Enteric adenoviruses (HAdV-40, HAdV-41) differ from other human serotypes by the presence of two fibers of different lengths on the virion.…”

“…Even if adenovirus dodecahedron's biological function is not fully understood so far, this versatile virus-like particle can be used for biotechnology applications [17][18][19][20] or as a tool for adenovirus receptor identification [8] . In order to gain further insight into the function of the enteric adenovirus SF, we constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base by coexpression in a baculovirus system.…”