Canine Adenovirus Type 2 Attachment and Internalization: Coxsackievirus-Adenovirus Receptor, Alternative Receptors, and an RGD-Independent Pathway

Abstract

The best-characterized receptors for adenoviruses (Ads) are the coxsackievirus-Ad receptor (CAR) and integrins ␣ v ␤ 5 and ␣ v ␤ 3 , which facilitate entry. The ␣ v integrins recognize an Arg-Gly-Asp (RGD) motif found in some extracellular matrix proteins and in the penton base in most human Ads. Using a canine adenovirus type 2 (CAV-2) vector, we found that CHO cells that express CAR but not wild-type CHO cells are susceptible to CAV-2 transduction. Cells expressing ␣ M ␤ 2 integrins or major histocompatibility complex class I (MHC-I) molecules but which do not express CAR were not transduced. Binding assays showed that CAV-2 attaches to a recombinant soluble form of CAR and that Ad type 5 (Ad5) fiber, penton base, and an anti-CAR antibody partially blocked attachment. Using fluorescently labeled CAV-2 particles, we found that in some cells nonpermissive for transduction, inhibition was at the point of internalization and not attachment. The transduction efficiency of CAV-2, which lacks an RGD motif, surprisingly mimicked that of Ad5 when tested in cells selectively expressing ␣ v ␤ 5 and ␣ v ␤ 3 integrins. Our results demonstrate that CAV-2 transduction is augmented by CAR and possibly by ␣ v ␤ 5 , though transduction can be CAR and ␣ v ␤ 3/5 independent but is ␣ M ␤ 2 , MHC-I, and RGD independent, demonstrating a transduction mechanism which is distinct from that of Ad2/5.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy

“…CAV-2 has been shown to bind directly to soluble recombinant human CAR and can utilize human or murine CAR for cell entry in vitro. 27 This finding, combined with our observation that both CAV-1 and CAV-2 knobs can competitively decrease Ad5-mediated gene transfer, suggest that this common receptor may likely be CAR.…”

“…Since CAV-2 has been shown to bind to CAR, 14,27 we investigated whether CAR may be a common receptor for the CAV-1 and CAV-2 knobs. For this reason, we performed a blocking experiment of Ad5Luc1-mediated gene transfer in U118-hCAR-tailless cells (a CAR-positive U118 cell line variant that heterelogously expresses the extracellular domain of human CAR 28 ) with CAV-1 and CAV-2 fiber-knob proteins.…”

“…18 The tropism of CAV-2 fiber-knob has also been applied in the context of a chimeric Ad5 vector, which provided significant infectivity enhancement over an unmodified Ad5 virus. 14 CAV-1, however, has not been investigated for gene therapy purposes and represents a potential basis for achieving novel adenoviral tropism.…”

“…Although some of these viruses are being developed as gene delivery vectors themselves, 16 the substitution of the Ad5 fiber-knob with these various xenotype fiber-knobs provides an efficient means to analyze their tropism in the context of an Ad5 vector that has been rigourously studied and for which molecular methods have been well established. 2 Previously, we exploited the unique tropism of canine adenovirus serotype 2 (CAV-2) 18 to generate an Ad5/ CAV-2 chimeric vector, which exhibited profound infectivity enhancement of CAR-deficient human tumor cell targets. 14 Another canine adenovirus strain, serotype 1 (CAV-1), has also been partially characterized.…”

L’adénovirus canin : meilleur ami de l’homme ?

“…En outre, le génome de CAV-2 ne s'intègre pas non plus à celui de la cellule hôte ce qui a l'avantage de minimiser le risque de transformation, mais entraîne aussi la perte d'expression du transgène dans des cellules en prolifération, une expression à long terme nécessitant donc, soit des ré-administrations du vecteur, soit le ciblage des cellules post-mitotiques. Enfin, si la majorité des Ad utilisent la protéine CAR (coxsackievirus adenovirus receptor) comme récepteur principal et certaines intégrines comme récepteur secondaire (activant des cascades de signalisation qui aboutissent à la production de cytokines pro-inflammatoires [4]), CAV-2 a l'avantage: (1) de ne pas posséder le motif responsable de l'interaction avec les intégrines [5]; et (2) de n'infecter que les cellules exprimant CAR [6], ce qui restreint son tropisme et pourrait permettre de cibler une population cellulaire au sein d'un tissu [7]. In vivo, les vecteurs CAV-2 transduisent efficacement les cellules épithéliales des voies aériennes, in vivo chez la souris ou in vitro chez l'homme [2], encourageant le développement de vecteurs CAV-2 pour traiter la mucoviscidose, maladie récalcitrante à la thérapie génique.…”