The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

Abstract

Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the in vivo gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2 cap sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids 588 QQNTA 592 of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process.

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

“…The biological force(s) responsible for driving the biophysical divergence between serotypes is unclear at this time. What is known is that sequence differences in the VRs contribute to phenotypic differences, including receptor attachment, transduction efficiency, and antigenic reactivity (1,3,4,88,90,91,95,96,105). We hypothesize that the observed biophysical differences between serotypes are connected to the specific biology of each serotype.…”

“…Our proteolytic map also overlies a recent cryo-EM reconstruction of AAV8 in complex with a neutralizing antibody, which highlights the 3-fold region as an important antigenic determinant as well (90). Residues on the 3-fold protrusions of AAV8 also determine receptor binding and transduction efficiency (19,90,91). Cleavage sites near the 5-fold axis did not show up in our maps, indicating that this region is not highly dynamic under the conditions use this region is dynamic under certain conditions, because conformational diversity is present in structural models (1, 3).…”

Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant ‐7m8

“…All rights reserved 15 has been identified as its co-receptor and AAV8 binding to lamR is mediated by the amino acids 491 to 547 and 593 to 623 (Akache et al, 2006). Moreover, peptide insertions on AAV8 capsid at position 590 allow modification of its tropism (Michelfelder et al, 2011;Raupp et al, 2012) but amino acids 588 to 592 are not necessary for AAV8 binding and uptake. This region is thus involved in but not necessary for interaction with cellular receptors (Raupp et al, 2012).…”

Structural Insights into Adeno-Associated Virus Serotype 5

“…The AAV capsid surface features that are unique to AAV5-shorter 3-fold protrusions, extended VR-VII, and smaller HI loop-contain amino acid residues or are proximate to capsid regions reported to play essential roles in the AAV life cycle. For example, residues within VR-IV, VR-V, and VR-VIII which make up the 3-fold protrusions have been reported to control glycan receptor attachment (VR-V and VR-VIII for AAV2 and VR-V for AAV9) (90)(91)(92)(93)122), transduction (VR-IV, VR-V, and VR-VIII in AAV2, VR-VIII in AAV8, and VR-IV, VR-V, and VR-VIII in AAV9) (94)(95)(96)(97)(98)(99) and antigenic phenotypes (VR-IV, VR-V, and VR-VIII in AAV2 and VR-VIII in AAV8 (97,100,101). These regions have similar roles in AAV5.…”