A common mechanism for cytoplasmic dynein-dependent microtubule binding shared among adeno-associated virus and adenovirus serotypes

Abstract

During infection, adenovirus-associated virus (AAV) undergoes microtubule-dependent retrograde transport as part of a program of vectorial transport of viral genome to the nucleus. A microtubule binding assay was used to evaluate the hypothesis that cytoplasmic dynein mediates AAV interaction with microtubules. Binding of AAV serotype 2 (AAV2) was enhanced in a nucleotide-dependent manner by the presence of total cellular microtubule-associated proteins (MAPs) but not cytoplasmic dynein-depleted MAPs. Excess AAV2 capsid protein prevented microtubule binding by AAV serotypes 2, 5, and rh.10, as well as adenovirus serotype 5, indicating that similar binding sites are used by these viruses.

FIG. 1.

Analysis of MAP-dependent AAV interaction with microtubules. To evaluate AAV2-microtubule interaction, a previously published microtubule binding assay was performed (15). Cy3-conjugated AAV2 was combined with MAP-containing or MAP-depleted microtubules from A549 cell lysate, incubated, and then centrifuged to pellet microtubules. Supernatant (S) and pellet (P) were then analyzed by gel electrophoresis. A fluorescence scan (top) and a Coomassie blue stained gel (bottom) show that MAPs enhance AAV2-microtubule interaction and pelleting.

FIG. 2.

Coimmunoprecipitation of cytoplasmic dynein, dynamitin, and AAV. Cy3-conjugated AAV2, AAV5, or AAVrh.10 capsids were added to cell lysate, incubated, and immunoprecipitated by using a monoclonal anticytoplasmic dynein antibody (15). Immunoprecipitated AAV proteins were analyzed by gel electrophoresis and fluorescence scanning. Cytoplasmic dynein and dynamitin were evaluated by immunoblotting. (A) Coimmunoprecipitation of AAV2 and the dynein complex. (B) Coimmunoprecipitation of AAV5 and the dynein complex. (C) Coimmunoprecipitation of AAVrh.10 and the dynein complex.

FIG. 3.

Common mechanism for microtubule interaction among divergent AAVs. MAP-containing microtubules were isolated from A549 cells as described above and incubated with an Sf9 cell lysate derived from naive Sf9 cells or from Sf9 cells infected with a baculovirus expressing the AAV2 cap gene (producing VP1, -2, and -3) or a null baculovirus. Cy3-AAV2, Cy3-AAV5, Cy3-AAVrh.10, or Cy3-adenovirus were incubated with the Sf9 lysates and pelleted. Supernatant (S) and pellet (P) were then analyzed by gel electrophoresis. (A) AAV2 fluorescence scan; (B) AAV5 fluorescence scan; (C) AAVrh.10 fluorescence scan; (D) adenovirus fluorescence scan. The Sf9 lysate containing AAV2 VP1, -2, and -3 inhibited virus-microtubule interaction, whereas control Sf9 cell lysates did not.