Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

Abstract

As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the ␤HI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus. IMPORTANCEDespite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild-type virus.

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

“…Structural alignment showed that AAV4 K492 and K503, which superpose with AAV1 S499 and T504, are adjacent to the SIA binding residue N500 and W503 on AAV1. AAV5 binds to SIA by M569, A570, T571, A581, G583, T584, Y585, N586, and L587 (65). However, none of these AAV5 residues is close to the SIA binding site on AAV1.…”

“…This MAb inhibits infection at a postnuclear entry step. For AAV5, mutation of its SIA binding site has also been shown to create variants able to escape from antibody recognition (65), again pointing to a neutralization mechanism involving receptor attachment blockage. In the example of AAV8, its ADK8 MAb blocks infection at a post-cell/prenuclear-entry step, suggesting a block in cellular trafficking (58).…”

Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

“…However, it is also possible that neutralization by ADK5b is due to the occlusion of residues on the wall of the 3-fold protrusion facing the 5-fold axis and/or the DE loop forming the 5-fold cylinder, which are unique to the epitope for this MAb. Significantly, the SIA A site has been implicated in SIA-dependent cellular transduction (77,80). It is possible that the binding of ADK5b to the outer wall of protrusions has an effect on this function.…”

“…It has been shown that AAV5 can utilize sialic acid (SIA) as a receptor to infect cells (74)(75)(76), and two SIA binding sites, A and B, have been described (77). The A site is at the center of the 3-fold axis and includes residues 569, 570, 571, and 583 to 587; the B site is located under the HI loop close to the 5-fold axis and includes residues 277, 279, 280, 350 to 354, 359, 361 to 363, 533, and 650 to 652.…”

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

“…Sialic acid is required for AAV5 binding and infection (Walters et al, 2004). Afione and collaborators have recently identified AAV5 sialic acid binding region (Afione et al, 2015),…”