Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

Abstract

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be important considerations for the rational design of improved gene therapy vectors.

Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

“…To validate that the AAV2 or -3 capsid structures defined as binding sites for natural heparin also mediate binding to synthetic heparin variants, AAV2 (R585A/ R588A) and AAV3 (R594A) capsid mutants that had been shown previously to be deficient for binding to natural heparin were generated (27,28). As anticipated, fluorophore-labeled AAV2 or AAV3 capsid mutants were unable to bind to any of the specific heparins (Fig.…”

Multiple Roles for Sialylated Glycans in Determining the Cardiopulmonary Tropism of Adeno-Associated Virus 4

“…The 3-fold symmetry axes on AAV capsids have been associated with glycan receptor recognition in the cases of AAV2, AAV3b, AAV6, and AAV9 (25)(26)(27)(28)(29)(30)(31). The surface protrusions and spikes surrounding the 3-fold axes are predominantly composed of the highly dynamic GH loop connecting the ␤G and ␤H strands within the capsid protein subunit (10,11).…”

“…For instance, key basic residues (R484, R487, K532, R585, and R588) involved in heparan sulfate recognition have now been located on 3-fold protrusions of the AAV2 capsid (25,27,29,41). Similarly, heparan sulfate recognition by AAV3B and AAV6 capsids is now well established (26,28,31). In the case of AAV9, the residues D271, N272, N470, Y446, and W503 are required for Gal binding (30).…”

Adeno-Associated Viral Vectors Based on Serotype 3b Use Components of the Fibroblast Growth Factor Receptor Signaling Complex for Efficient Transduction

“…Initially deposited into the American Type Culture Collection (ATCC, Manassas, VA) as AAV3 strain H (VR-681; ATCC), two distinct AAV3 isolates (AAV3a and AAV3b) differing by only six amino acids have been subsequently cloned (Muramatsu et al, 1996;Rutledge et al, 1998). Both appear to bind to heparin and the infectivity of both is inhibited by soluble heparin, albeit at much higher doses than for AAV2 (Handa et al, 2000;Rabinowitz et al, 2004;Lerch and Chapman, 2012). In comparison with vectors based on other AAV serotypes, AAV3 vectors inefficiently transduce most cell types with the exceptions of cochlear inner hair cells (Chang et al, 2005) and human liver cancer cells (Glushakova et al, 2009).…”

“…This is believed to occur because the AAV3b capsid interacts with less affinity to heparin than AAV2 (Rabinowitz et al, 2002;Lerch and Chapman, 2012), a trait ascribed to its lack of highaffinity heparin-binding sites that exist on the closely related AAV2 capsid (Opie et al, 2003).…”