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. 2003 Oct;77(20):11072-81.
doi: 10.1128/jvi.77.20.11072-11081.2003.

Identification of a heparin-binding motif on adeno-associated virus type 2 capsids

A Kern  1 , K Schmidt, C Leder, O J Müller, C E Wobus, K Bettinger, C W Von der Lieth, J A King, J A Kleinschmidt
Affiliations

Identification of a heparin-binding motif on adeno-associated virus type 2 capsids

A Kern et al. J Virol. 2003 Oct.

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.

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Figures

FIG. 1.
FIG. 1.
Amino acids of AAV-2 VPs subjected to site-directed mutagenesis. Blockwise conversions of VP sequences (VP1 numbering) of AAV-2 to those of AAV-3 (mutants 1, 2, 3, 4, and 5) are highlighted by grey rectangles. Single mutated amino acids are highlighted by grey circles. Mutants are numbered as shown in Table 1.
FIG. 2.
FIG. 2.
Influence of mutations of basic amino acids in loop IV on infectivity, cell binding, and heparin binding of AAV-2. Virus stocks were produced by transfection of wild-type (wt) or mutated AAV-2 genomic plasmids into 293T cells and superinfection with adenovirus (multiplicity of infection, 50). (a) Analysis of arginines 585 and 588 for involvement in heparin binding. (b) Analysis of distantly located basic amino acids predicted to possibly contribute to heparin binding. (c) Further loop IV basic amino acids involved in heparin binding. Infectious units were determined as described in Materials and Methods. Capsid titers and genomic titers were not influenced by the mutations (data not shown), implying that they did not affect capsid assembly or genome encapsidation. Virus bound to HeLa cells was measured with monoclonal antibody A20 (68). Heparin binding was assayed by chromatography of virus preparations (freeze-thaw lysates) with heparin-agarose and quantitation of applied, flowthrough, and salt-eluted virus with the A20 capsid ELISA. The amount of applied virus was set to 100%. Error bars indicate standard deviations of at least three independent experiments.
FIG. 3.
FIG. 3.
In vivo distribution of wild-type rAAV and rAAV mutant R484E/R585E in mouse tissues. (A) Luciferase activities in different organs after intravenous injection of 1011 wild-type capsids or rAAV mutated in R484E and R585E 3 weeks postinfecion. Luciferase activities are depicted in relative light units (RLU) per milligram of protein. Error bars indicate standard deviations. (B) Detection of luciferase gene transfer by PCR in selected organs 3 weeks after intravenous infusion of 1011 vector genomes of rAAV mutant R484E/R585E and wild-type rAAV. Amplification of β-actin was used as a control. The 677-bp luciferase band is barely detectable in liver tissues of mice injected with double mutant R484E/R585E but is clearly visible in mice injected with wild-type rAAV. PCR amplification of tissue samples from heart, lungs (data not shown), and kidneys (data not shown) did not reveal any differences in luciferase gene transduction.
FIG. 4.
FIG. 4.
Localization of amino acids involved in heparin binding on the AAV-2 capsid surface. (A) The amino acids involved in heparin binding—R585 (orange), R588 (red), R484 (purple), R487 (green),K532 (blue), and R475 (yellow)—are grouped in three clusters at the threefold spike region of the capsid. The inset shows a VP trimer which forms the threefold spikes. (B) A side view of a VP trimer shows the surface exposure of the basic clusters at the inner shoulder of the threefold spikes and along a channel-like structure between the threefold spikes. R475 is buried in the capsid wall (at the border of two subunits [not shown]) and likely affects heparin binding indirectly when it is mutated to alanine. The unmarked residues are derived from the second and third spikes. (C) Two different subunits of a VP trimer (indicated in red, blue, and green) contribute to the formation of a basic heparin-binding cluster. R585 and R588 of the red, green, or blue subunit (shown as orbitals, with the red subunit labeled) are surrounded by K532, R484, and R487 of another subunit (orbitals of the red subunit which surround R585 and R588 of the blue subunit are indicated by arrows).
FIG. 5.
FIG. 5.
Computer simulation of heparin docking to the AAV-2 capsid surface. The docking simulation was performed as described in Materials and Methods. The light blue orbitals show the heparin hexamer. Amino acids which are in proximity to the heparin molecule and which might permit electrostatic or hydrogen bond interactions are indicated. Letters A, B, and C indicate the three different VP subunits involved in heparin binding.
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