Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids

Abstract

Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin-and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…For controls, recombinant vectors packaged into non-targeted wt AAV2 capsids (wt rAAV2), into a heparin binding-deficient AAV2 capsid (rAAV2-R484E; R585E), 26,38 or into AAV9 capsids (rAAV2/9), were generated expressing the firefly luciferase reporter gene. Considering the experimental variation in treating individual animals, we compared in vivo gene delivery profiles between targeted viruses and non-targeted vectors upon co-injection of both into the same animal ( Figure 3a).…”

“…In earlier studies, rAAV2-R484E;R585E showed strongly reduced transduction of mouse hepatic tissue in vivo, with slight increase in heart transduction. 26,38 Thus, this experiment should clarify whether the ablation of heparin binding alone-which may result from insertion of a peptide at position R588-would result in similarly increased heart targeting (Figures 3b [4] and [5] and c [4] and [5]). Mutation of the heparin-binding motifs indeed slightly increased heart infection and dramatically reduced liver transduction.…”

“…[8][9][10][17][18][19] Whereas larger peptides up to the size of GFP (238 amino acids) could be accommodated at the VP2 N-terminus 15,[20][21][22][23][24][25] the addition of small peptides up to 34 amino acids is tolerated at several positions of the AAV2 capsid. 1 Among the tolerated insertion sites for small peptides, insertions in the AAV2 heparin binding domain 26,27 adjacent to amino acids 587 or 588 was most successful. 17,23,[28][29][30][31][32] An inherent problem of genetic modification of the AAV capsid is the design of the targeting peptide.…”

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity

“…Briefly, AAV2 and AAV6 capsids normally bind heparin [13]. AAV2 contains an RXXR consensus sequence (RGNR) on VP3 that mediates heparin binding [16,23]. AAVhu29R is a variant of AAV2 capsid with SGNT at the RXXR sequence that blocks heparin binding, in addition to five other single amino acid changes outside the RXXR region [30].…”

Diabetes enhances the efficacy of AAV2 vectors in the retina: therapeutic effect of AAV2 encoding vasoinhibin and soluble VEGF receptor 1

“…54 HSPG associated with cell membranes (syndecans and glypicans) serve as primary receptors, enabling vector docking and the subsequent interactions with AAV2 co-receptors (FGFR1, αvβ5, and HGFR) required for endosomal entry. Also, HSPG associated with the extracellular matrix (perlecans) help store and concentrate AAV2, thus enabling their presentation to attachment and entry receptors.…”