Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6

Abstract

The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7-and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (␤B to ␤I) ␤-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.

Structural Insights into Adeno-Associated Virus Serotype 5

“…Unlike the structures determined for some other AAV capsids, for example, AAV3B, AAV4, AAV6, and AAV8, including VLPs produced in baculovirus/Sf9 expression systems (e.g., AAV6 and AAV8), where at least one DNA nucleotide is ordered (22,23,26,27,29), there was no DNA ordered in the AAV5 capsid interior in the previously identified DNA binding pocket, despite the conservation of the binding pocket amino acids. The lack of ordering of a nucleotide in AAV5 is predicted to be due to the high radiation sensitivity of the thin crystals used for X-ray diffraction data collection.…”

“…An additional visual comparison of the superposition of the structures was carried out using the Coot program (62). The available crystal structures of AAV3b, AAV6, AAV8, and AAV9 (23,26,27,29) were not included in this analysis due to their high similarity to AAV2 and the fact that regions which vary between them and AAV2 were already described when this serotype was compared to AAV4.…”

“…Structures determined for AAVs show capsids assembled from the common VP3 region (20)(21)(22)(23)(24)(25)(26)(27)(28)(29). There is currently no experimentally determined structure for VP1u or the overlapping VP1/ VP2 N terminus.…”

The AAV9 receptor and its modification to improve in vivo lung gene transfer in mice

“…The baculovirus/Sf9 protein expression system provides a tractable method for the production of large quantities of AAV virus-like particles (VLPs) shown to be structurally and antigenically similar to virus particles and virions produced in mammalian cell culture systems (34,35). For GMA binding studies, VLPs of AAV9 were prepared using the Bac-to-Bac baculovirus/Sf9 expression system (Invitrogen) as previously described (36) and screened in a high-throughput GMA developed by Cores D and H of the Consortium for Functional Glycomics (CFG; an NIH National Institute of General Medicine Science Initiative; ref.…”

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

“…A commonality of all AAV structures determined to date is regions of protein/DNA interaction with a well-ordered nucleotide. These interactions are present in capsids packaging heterologous cellular DNA or specific viral sequences (27,43,44,47).…”