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 an important consideration for the rational design of improved gene therapy vectors.

Figure 1

Identification of potential heparin binding residues on AAV-3B. Two views near the 3-fold promixal spikes of AAV-3B are shown. (A) The spike-like protrusions of AAV-3B viewed down a 3-fold symmetry axis. A ribbon representation of the AAV-3B capsid can be seen beneath the translucent molecular surface, which is colored by electrostatic surface potential (blue = positive; red = negative). Two regions near residues Arg₄₄₇ and Arg₅₉₄ have strong positive surface charge and were identified as candidate receptor binding sites. (B) Structural overlay of a single spike from AAV-3B (green) and AAV-2 (magenta). Arg₄₄₇ is conserved in AAV-2, but forms a salt bridge with Glu₄₉₉ (dashed line). Asn₅₀₀ is the equivalent residue in AAV-3B and, as a neutral amino acid, does not pair with Arg₄₄₇, leaving a stronger positive surface charge at this site. On the left, differences in the AAV-2 HSPG site are highlighted with Arg₅₈₅ & Arg₅₈₈ of AAV-2 replaced by Ser₅₈₆ and Thr₅₈₉ in AAV-3B.

Figure 2

The SOS binding site on AAV-3B. Difference (mFo-DFc) electron density (blue mesh, contoured at 5σ) indicates SOS bound where it can interact electrostatically with Arg₅₉₄ in maps calculated using data from the AAV-3B:SOS co-crystals. The density can accommodate a single SOS molecule bound to the capsid on the 3-fold symmetry axis. A ribbon representation of the 2.6Å AAV-3B structure (Lerch, Xie, and Chapman, 2010) is overlayed with a translucent molecular surface.

Figure 3

Heparin-affinity chromatography for AAV-3B mutants designed for diminished binding. Samples were applied to a heparin column, washed, and eluted in PBS with increasing NaCl concentrations. Capsids were assayed by ELISA, using a capsid-specific monoclonal antibody (Grimm et al., 1999). (A) Capsids were detected in the flow-through and 1^st wash fraction only for AAV-3B mutants R594A and R594E, while all other capsids retained at least some affinity. (B) Elution profiles. The [NaCl] added is shown, and the total [NaCl], including the 137mM of the PBS running buffer, is shown in parentheses. AAV-3B bound most tightly, and N500E and R447A eluted in slightly lower NaCl concentrations, suggesting that these residues might play a minor role in heparin binding. R594A or R594E capsid mutants were not detected in any of the elution fractions, indicating that mutation of Arg₅₉₄ abrogates heparin binding by AAV-3B.

Figure 4

Heparin affinity chromatography of AAV-3B mutants with enhanced binding. The heparin binding residues from AAV-2 (Arg₅₈₅ & Arg₅₈₈) were introduced into the equivalent positions in AAV-3B (replacing Ser₅₈₆ and Thr₅₈₉). (A) The heparin binding affinity of the single S586R and T589R mutants was increased over that of AAV-3B, and was comparable to that of AAV-2. AAV-3B S586R/T589R shows a striking increase in heparin binding affinity, presumably due to the combination of heparin binding sites from two serotypes. (B) AAV-3B S586R/T589R, N500E/S586R/T589R and R447A/S586R/T589R mutants all bound heparin with high affinity, while R594A/S586R/T589R showed weaker binding, comparable to that of AAV-2.

Figure 5

Cell binding by AAV-3B mutants. AAV capsids bound to HeLa cells were detected by a cell-based ELISA (Kern et al., 2003). (A) AAV-2 capsids bind cells to a greater extent than WT AAV-3B at similar concentrations. (B) Comparison of cell attachment for the enhanced-affinity AAV-3B mutants. S586R/T589R, N500E/S586R/T589R and R447A/S586R/T589R all bound cells comparably and at higher levels than AAV-2. The R594A/S586R/T589R mutant, however, showed decreased cell binding over the S586R/T589R mutant. As observed for heparin, R594A/S586R/T589R bound cells at levels comparable to AAV-2.

Figure 6

Arg₅₉₄ is for a key determinant of AAV-3B transduction. HeLa cells (shown at 200x magnification) were treated with WT or R594A AAV-3B vectors carrying a GFP gene. GFP expression was not observed in control cells (A), whereas strong expression was observed in cells transduced with AAV-3B vectors (B). AAV-3B R594A vectors did not transduce cells at 250 vg/cell (C), but GFP expression was observed in a few cells after treatment with 1000 vg/cell (D, arrow).