Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site

Abstract

The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wildtype AAV6. Furthermore, three of the AAV1 SIA binding site mutants-S472R, V473D, and N500E-escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface "hot spot" dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering. IMPORTANCEThe adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and the detrimental effects of preexisting host antibodies. This study aimed to identify key capsid regions which can be engineered to overcome these limitations. A sialic glycan receptor recognition pocket was identified in AAV1 and its closely related AAV6, using X-ray crystallography. The site was confirmed by mutagenesis followed by cell binding and transduction assays. Significantly, residues controlling gene expression efficiency, as well as antibody escape variants, were also identified. This study thus provides, at the amino acid level, information for rational structural engineering of AAV vectors with improved therapeutic efficacy.

Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

“…Specifically, AAV1 requires sialylated glycans for cell surface attachment, which in turn determines transduction efficiency (23). The SA-binding site of AAV1 was resolved recently, revealing specific contact residues located in a pocket at the base of the threefold protrusions similar to the galactose binding site on AAV9 (16). Specific residues located within the SA binding pocket include S268, D270, N271, N447, S472, V473, N500, T502, and W503.…”

“…Here we tested this hypothesis using AAV1 as a template, which we chose because of the availability of extensive structural information pertaining to this capsid complexed with different mAbs and its cognate glycan receptor, sialic acid (SA) (9,10,16). We then combined structural information from cryo-EM images of AAV1 capsids complexed with three different murine mAbs and directed evolution without selective pressure from NAbs to generate antibodyevading AAV variants.…”

Recombinant Adeno-Associated Virus Serotype 6 (rAAV6) Potently and Preferentially Transduces Rat Astrocytes In vitro and In vivo

“…Although we did not pursue further the molecular underpinnings for this phenomenon, some inferences may be made based on the already existing literature. Several studies found that AAV1 and AAV6, which have structurally similar (∼99% homologous) capsid proteins, use α2,3 and α2,6 N -linked sialic acid moieties of the cell surface proteoglycans for entry (Wu et al, 2006c; Huang et al, 2016). In the present work, we found a strong dissimilarity between rAAV1 and rAAV6 in terms of their efficacy to transduce primary rat astrocytes in cell culture.…”

Structural features of glycan recognition among viral pathogens

“…GAGs are long unbranched polysaccharides composed of repeating disaccharide units of sulfated uronic acid and glucosamine residues (Figure 4c), and includes heparan sulfate (HS). Specific recognition of GAGs for cell attachment have been implicated for a number of non-enveloped icosahedral viruses such as human papillomavirus (HPV), belonging to Papovaviridae [48]; human parvovirus and adeno-associated viruses (AAV), belonging to Parvovirida e [9,49]; and enveloped icosahedral viruses such as Zika virus, Dengue virus and West Nile virus, belonging to Flaviviridae [50]. Although structural studies have provided general features of how these viruses interact with the negatively-charged HS, detailed understanding of the rules of glycan engagement is limited due to lack of atomic-level characterization of glycan interactions.…”