Specific binding of the adenovirus capsid to the nuclear envelope.
In Vitro Techniques
Adenovirus (Ad) vectors used for gene therapy are efficient in entering the infected cell and targeting their genome to the nucleus. To study the mechanism of the interaction between Ad and the nuclear envelope we have established an in vitro assay using rat liver nuclei incubated with serotype 5 Ad vector. Binding of either fluorophore (Cy3)-labeled Ad5 (Cy3-AdGFP) or [3H]Ad5 was blocked by excess unlabeled Ad5, indicating that the interaction was specific. Binding reached equilibrium within 30 min, exhibited temperature dependence with more binding occurring at 37 degrees C than at 4 degrees C and appeared to be irreversible. Prior treatment of nuclei with glutaraldehyde or proteolysis of nuclei with trypsin inhibited the Cy3-AdGFP association with nuclei, and pretreatment of Cy3-AdGFP with human anti-Ad5 serum, but not naive human serum, inhibited Cy3-AdGFP, suggesting a requirement for direct interaction between Ad5 and nuclei. Addition of excess unlabeled Ad serotype 2 or Ad serotype 7 competed for binding with Cy3-AdGFP, indicating that the capsid determinant of nuclear binding was conserved among group B and C Ad serotypes. These data suggest that the Ad capsid and nuclear envelope contain specific domains that mediate binding of the two entities and that binding mechanisms to the nuclear envelope might be a common final pathway of different Ad serotypes.