J. as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence KW-2449 for each bound Fab around the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for computer virus neutralization by the antibodies. INTRODUCTION Antibodies that are elicited against computer virus capsids represent a critical component of the host protective response in vertebrates. For most viruses, they control both the susceptibility of an animal to contamination and also the recovery from disease. For human gene delivery, the presence of preexisting antibodies or antibodies that develop after administration of viral vectors can create significant complications for the application or reapplication of therapies (1C4). The host antibody responses initiate through the binding and activation of B cells Mouse monoclonal to CD95(Biotin) and are originally composed of low-affinity IgM variants; the B cells are subsequently selected for enrichment of higher-affinity antibody variants, which class-switch to form IgG1 and other subtypes. However, details of the production of effective immune responses against viral antigens and the structural features of epitopes on viruses are still only partially understood (5C8). Adeno-associated viruses (AAVs) consist of a T=1 icosahedral capsid composed of three related, overlapping viral structural proteins (VP1, VP2, and VP3), which differ in their N termini, while the unique N-terminal region of VP1 (VP1u) is essential for capsid trafficking within the cell during infection (9C12). VP3 is contained entirely within the sequence of VP2, which is, in turn, contained within VP1. In KW-2449 the three-dimensional (3D) structures of AAVs determined thus far, only the 520 amino acids (aa) within the VP3 common region have been observed (13C17). VP3 contains an eight-stranded -barrel core, with the -strands linked by extended loops that form the capsid surface (Fig. 1A). These loops, the largest of which is the GH loop (230 aa) located between the G and H strands, also contain stretches of -strand structure (Fig. 1B). The loops exhibit the highest sequence and structural variation in the VP3 KW-2449 region and contain nine structurally variable regions (VRs; VR-I to VR-IX) (defined in reference 14) (Fig. 1A and ?andB),B), which have roles in receptor attachment, tissue transduction, and antigenicity (reviewed in references 14, 17, 18, 19, and 20). The AAV capsid surface topology (Fig. 1A) is characterized by prominent features, such as depressions at the icosahedral 2-fold axis and around a channel-like structure at the 5-fold axis and protrusions that surround each icosahedral 3-fold axis. The depressions vary in width, while the protrusions vary in width and height among different AAVs (14, 15). Open in a separate window Fig 1 Variable regions on the AAV capsid surface. (A) Ribbon diagram (left) of an AAV2 VP3 monomer highlights the eight -strands that make up the core -barrel (gray ribbon) and loops inserted between the strands that make up the capsid surface. The nine AAV variable regions (VR-I to -IX) (defined in reference 14) are colored as follows: I, purple; II, blue; III, yellow; IV, red; V, black; VI, hot pink; VII, cyan; VIII, green; and IX, chocolate. The two -sheets of the -barrel are indicated by arrows, and the A helix, the DE (between D and E) and HI (between H and I) loops, and N and KW-2449 C termini KW-2449 are labeled. The approximate positions of the 2-, 3-, and 5-fold axes are indicated by filled oval, triangle, and pentagon symbols, respectively. At right is an AAV2 capsid (gray; assembled from 60 VP3 monomers) showing the location of the VRs, colored as described for panel A, with the HI loop shown in wheat. The.