The Adeno-Associated Virus Crystal: Impact Inversely Proportional to Size
Authors: Joseph E. Rabinowitz, R.Jude Samulski
Journal: Molecular Therapy (2002)

Michael Chapman and his team at Florida State University have created a detailed structural map of adeno-associated virus serotype 2 (AAV2) at 3 A resolution by X-ray crystallography [1Xie Q. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.Proc. Natl. Acad. Sci. USA. 2002; 99: 10405-10410Crossref PubMed Scopus (459) Google Scholar]. AAV2 and the other AAV serotypes are promising vectors for gene transfer in human gene therapy protocols. The crystal structure of AAV2 will provide the crucial knowledge to decipher the existing mutagenesis data and to direct all future retargeting modifications of AAV2 and the other serotypes. The parvoviruses (parvo is Greek for small) are a large family of approximately 25-nm diameter, non-enveloped viruses with icosahedral symmetry. AAV2 is a nonpathogenic human parvovirus classified as one of the “dependo” viruses because it requires helper viruses to complete its lytic life cycle [2Muzyczka, N., and Berns, K.2001. Parvoviridae: the viruses and their replication. In VirologyB. Fields, D. Knipe, and M. Howley, Eds., pp. 2327–2369. Lippincott, Philadelphia.Google Scholar]. AAV2 is the first dependovirus crystal structure to be determined. Previously, genetic modification of AAV2 capsids has relied on random mutagenesis [3Rabinowitz J. Xiao X. Samulski R. Insertional mutagenesis of AAV2 capsid and the production of recombinant virus.Virology. 1999; 265: 274-285Crossref PubMed Scopus (106) Google Scholar], alanine scanning mutagenesis [4Wu P. Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.J. Virol. 2000; 74: 8635-8647Crossref PubMed Scopus (320) Google Scholar], and site-directed insertional mutations [4Wu P. 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Ther. 2001; 3: 964-975Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar] (crystal structure already determined [7Tsao J. The three-dimensional structure of canine parvovirus and its functional implications.Science. 1991; 251: 1456-1464Crossref PubMed Scopus (399) Google Scholar]) as a general guide to positioning insertions of foreign epitopes [4Wu P. Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.J. Virol. 2000; 74: 8635-8647Crossref PubMed Scopus (320) Google Scholar, 5Girod A. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.Nat. Med. 1999; 5: 1052-1056Crossref PubMed Scopus (22) Google Scholar, 6Grifman M. Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids.Mol. Ther. 2001; 3: 964-975Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar]. These approaches have determined some surface-displayed domains and regions that interfere with primary receptor binding. Other approaches have also yielded data on surface display, using overlapping peptides of AAV2 Vp1 capsid to determine which peptides react to neutralizing antibodies [8Moskalenko M. Epitope mapping of human anti-adeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure.J. Virol. 2000; 74: 1761-1766Crossref PubMed Scopus (208) Google Scholar] and using phage to display fragments of AAV2 capsid to identify epitopes involved in monoclonal antibody interactions [9Wobus C. Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection.J. Virol. 2000; 74: 9281-9293Crossref PubMed Scopus (225) Google Scholar]. The detail generated by the crystal structure has begun to integrate genetic data from epitope mapping and mutagenesis studies into a coherent picture—function has now been mapped to form. Xie et al. [1Xie Q. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.Proc. Natl. Acad. Sci. USA. 2002; 99: 10405-10410Crossref PubMed Scopus (459) Google Scholar] also noted the difference in topology between AAV2 and the other crystallized parvoviruses. There are striking similarities and differences between these crystal structures. The core of all parvoviruses shares the β-barrel motif, consisting of anti-parallel β-sheets. The sequence identity of the motif between autonomous and dependoviruses is low, although these motifs can be superimposed, suggesting a high degree of functional similarity. In contrast, the β-sheets that make up the barrel motif of AAV serotypes all share a high level of sequence identity. The looped-out domains between the β-sheets generally have less overall identity, with the longest loop located between β-sheets G and H (GH loop). This loop is approximately 230 amino acids in length for the AAV serotypes and many autonomous parvoviruses [10Chapman M. Rossmann M. Structure, sequence, and function correlations among parvoviruses.Virology. 1993; 194: 491-508Crossref PubMed Scopus (172) Google Scholar], and occupies greater than one-third of the total structure. The size of this loop, although similar between AAV serotypes and autonomous parvoviruses, is the most divergent of any sequence in the capsid. This low homology at the amino acid level is reflected in surface topology differences and in epitopes for receptor binding. AAV2 has three clusters of three peaks centered about the 3-fold axis of symmetry (Fig. 1, arrow). The peaks are not derived from one capsid subunit protein but from the interaction of two adjacent subunits. These peaks create a pocket with a diameter of 20 Å. The clusters of peaks are separated by 70 Å about the 3-fold axis of symmetry. The sequences that compose these structures are all from the GH loop. Cryo-electron microscopy of Aleutian mink disease parvovirus [11McKenna R. Three-dimensional structure of Aleutian Mink disease parvovirus: Implications for disease pathogenicity.J. Virol. 1999; 73: 6882-6891PubMed Google Scholar] and AAV2 [20Kronenberg S. Kleinschmidt J. Bottcher B. Electron cryo-microscopy and image reconstruction of adeno-associated virus type 2 empty capsids.EMBO Rep. 2001; 2: 997-1002Crossref PubMed Scopus (110) Google Scholar] shows similar clusters about the 3-fold axis, whereas CPV has a large spike at the 3-fold axis [7Tsao J. The three-dimensional structure of canine parvovirus and its functional implications.Science. 1991; 251: 1456-1464Crossref PubMed Scopus (399) Google Scholar, 10Chapman M. Rossmann M. Structure, sequence, and function correlations among parvoviruses.Virology. 1993; 194: 491-508Crossref PubMed Scopus (172) Google Scholar, 12Xie Q. Chapman M. Canine parvovirus capsid structure, analyzed at 2.9 A resolution.J. Mol. Biol. 1996; 264: 497-520Crossref PubMed Scopus (108) Google Scholar]. Comparatively, the insect densovirus, which has 96 fewer amino acids in its GH loop, has few surface features at or proximal to the 3-fold axis of symmetry [13Simpson A. Chipman P. Baker T. Tijssen P. Rossmann M. The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 A resolution.Structure. 1998; 6: 1355-1367Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar]. These examples illustrate the flexibility of the GH loop assembly. Based on this information, a flurry of genetic modifications centered on the GH-loop will be conducted to better understand viral structure/function domains. Similar studies of the adenovirus modification of the HI loop resulted in retargeting of adenoviral vectors. The interactions between capsid proteins at the 3-fold axis potentially play a crucial role during subunit assembly. In CPV, glycine residues at the base of these loops may allow for flexibility so loops from adjacent capsid proteins can fold over upon themselves [12Xie Q. Chapman M. Canine parvovirus capsid structure, analyzed at 2.9 A resolution.J. Mol. Biol. 1996; 264: 497-520Crossref PubMed Scopus (108) Google Scholar]. The thermodynamic benefit in terms of decrease in free energy suggests that assembly of CPV goes through a trimer intermediate [14Wu H. Rossmann M. The canine parvovirus empty capsid structure.J. Mol. Biol. 1993; 233: 231-244Crossref PubMed Scopus (81) Google Scholar]. Nevertheless, for AAV2, the loop structure BGH2 and BGH12-13 extend towards the adjacent subunits sandwiching the BGH6-7 of that adjecent subunit (Fig. 1). Additionally, Xie et al. [1Xie Q. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy.Proc. Natl. Acad. Sci. USA. 2002; 99: 10405-10410Crossref PubMed Scopus (459) Google Scholar] suggest that interactions at the 5-fold axis are potentially driving assembly. Regardless of how the assembly process of AAV2 is resolved, the crystal structure suggests specific experimental tests to elucidate these processes. The virion of AAV2, like all parvoviruses, is composed of more than one capsid protein, with the smallest of these proteins (Vp3) being the most abundant. Its sequence is completely contained within the sequence of the larger capsid proteins (Vp1 and Vp2). This creates an incomplete resolution of Vp1 and Vp2 non-overlapping sequences in the crystal structure. For AAV2, the largest capsid protein, Vp1, is 735 amino acids in length, yet only the carboxy-terminal 519 amino acids were clearly traceable. With this caveat, it should be stated that genetic and infectivity data have demonstrated that Vp3-only viruses bind heparin and compete with wild-type virus in transduction assays, but are noninfectious themselves [15Rabinowitz J. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity.J. Virol. 2002; 76: 791-801Crossref PubMed Scopus (625) Google Scholar]. The lack of infectivity may be due to lack of the phospholipase activity previously mapped to the Vp1 non-overlapping domain [16Zadori Z. A viral phospholipase A2 is required for parvovirus infectivity.Dev. Cell. 2001; 1: 291-302Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar], and shown to be essential for AAV infectivity. These data demonstrate the importance and interdependence of the genetic and crystallographic data in resolving differences in the structure and biology of AAV2. In the capsid sequence of AAV serotypes there are subdomains in the GH-loop that have little sequence identity, for example amino acids 443–475 (Vp1 numbering). This sequence includes the β-sheets GH2-GH3 and the most extended sequences from the center of the virus (Fig. 1, arrows). The amino acids between serine 452 and threonine 454 should be ideal for positioning an epitope for surface display. Another region that would be ideal for surface display is between threonine 491 and 503. This site has also been implicated in binding the neutralizing monoclonal antibody C37 [9Wobus C. Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection.J. Virol. 2000; 74: 9281-9293Crossref PubMed Scopus (225) Google Scholar]. Placing retargeting epitopes between these amino acids should disrupt an endogenous antigenic epitope and make this site important for developing novel AAV vaccine vectors. The crystal structure of AAV2 has brought consistency to the genetic data and has topologically mapped many important functions including primary receptor binding to the GH loop. Many studies have isolated mutations in the capsid that do not bind heparin sulfate [3Rabinowitz J. Xiao X. Samulski R. Insertional mutagenesis of AAV2 capsid and the production of recombinant virus.Virology. 1999; 265: 274-285Crossref PubMed Scopus (106) Google Scholar, 4Wu P. Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.J. Virol. 2000; 74: 8635-8647Crossref PubMed Scopus (320) Google Scholar, 5Girod A. Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.Nat. Med. 1999; 5: 1052-1056Crossref PubMed Scopus (22) Google Scholar], yet these mutations are spaced between amino acids 509 and 591 in the GH loop (Fig. 1, red). We have learned how these mutations fit into a coherent picture, and why the linear motif generally associated with heparin binding was not found in the Vp3 subunit. Basic amino acids line the region between the clusters of peaks, centering the basic residues about the 3-fold axis of symmetry [11McKenna R. Three-dimensional structure of Aleutian Mink disease parvovirus: Implications for disease pathogenicity.J. Virol. 1999; 73: 6882-6891PubMed Google Scholar] and implying that three subunits may be required for the interaction between AAV2 and heparin sulfate. Mixing genetically modified helper plasmids mutant for heparin binding with wild-type helper plasmids at varying ratios during virus production requires that two-thirds of the mixture be derived from the wild-type helper before normal heparin binding is observed (unpublished data). This observation lends support to multiple subunits interacting together to form a functional receptor binding site. However, the major implication in determining the amino acids responsible for heparin binding is the ability to ablate normal binding. The coupling of ablation with retargeting epitopes will have a great impact on the field of targeted gene therapy, and the crystal structure will allow this goal to be accomplished in a more-refined, directed manner. The crystal structure of adenovirus type 5 fiber represents a turning point in the development of retargeting vectors [17Xia D. Lynda H. Gerard R. Deisenhofer J. Crystal structure of the receptor-binding domain of adenovirus type 5 fiber protein at 1.7 A resolution.Structure. 1994; 2: 1259-1270Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar]. Although the HI loop was first thought to be a candidate for binding the cellular receptor, subsequent mutagenesis and structural studies have established the importance of the interaction of the AB loops with the CAR receptor [18Roelvink R. Mi lee G. Einfeld D. Kovesdi E. Wickham T. Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae.Science. 1999; 286: 1568-1571Crossref PubMed Scopus (335) Google Scholar, 19Bewley M. Springer K. Zhang Y. Freimuth P. Flanagan J. Structural analysis of the mechanism of adenovirus binding to its human cellular receptor, CAR.Science. 1999; 286: 1579-1583Crossref PubMed Scopus (373) Google Scholar]. Since these studies, research has focused on several areas, including generation of pseudotyped Ad, substitution of domains in the HI loop, ablation of the CAR binding epitope coupled with substitutions in the HI loop, removal of both CAR and integrin binding epitopes, and complete replacement of the fiber with exogenous sequence to develop artificial receptor molecules. The ability to learn from the examples of Ad retargeting will greatly facilitate the use of AAV vectors by ablation of endogenous tropism coupled with rational methods of retargeting. Many potential retargeting platforms can be envisioned by a combination of site-directed mutagenesis, insertional mutagenesis, serotype domain swaps, parvoviral domain swaps, and shuffling within the GH loop domains. Additionally, regions of opposing peaks lining the pocket of each 3-fold cluster can be exchanged with antibody binding sites or immune cell recognition sequences. The atomic structure of AAV2 represents a major step in rationally developing this vector and the other serotypes into the safest targeted long-term expressing gene therapy tool. The next several years should be quite exciting, as researchers begin to use this small virus in a big way.