Structurally Mapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4

Abstract

The adeno-associated viruses (AAVs) can package and deliver foreign DNA into cells for corrective gene delivery applications. The AAV serotypes have distinct cell binding, transduction, and antigenic characteristics that have been shown to be dictated by the capsid viral protein (VP) sequence. To understand the contribution of capsid structure to these properties, we have determined the crystal structure of AAV serotype 4 (AAV4), one of the most diverse serotypes with respect to capsid protein sequence and antigenic reactivity. Structural comparison of AAV4 to AAV2 shows conservation of the core ␤ strands (␤B to ␤I) and helical (␣A) secondary structure elements, which also exist in all other known parvovirus structures. However, surface loop variations (I to IX), some containing compensating structural insertions and deletions in adjacent regions, result in local topological differences on the capsid surface. These include AAV4 having a deeper twofold depression, wider and rounder protrusions surrounding the threefold axes, and a different topology at the top of the fivefold channel from that of AAV2. Also, the previously observed "valleys" between the threefold protrusions, containing AAV2's heparin binding residues, are narrower in AAV4. The observed differences in loop topologies at subunit interfaces are consistent with the inability of AAV2 and AAV4 VPs to combine for mosaic capsid formation in efforts to engineer novel tropisms. Significantly, all of the surface loop variations are associated with amino acids reported to affect receptor recognition, transduction, and anticapsid antibody reactivity for AAV2. This observation suggests that these capsid regions may also play similar roles in the other AAV serotypes.Adeno-associated viruses (AAVs), which are members of the Dependovirus genus of the Parvoviridae, are helper-dependent parvoviruses isolated from a number of different species, including humans (50). Despite a requirement for coinfection with a helper virus, such as adenovirus or herpesvirus, for productive replication, AAV capsids are similar to those of the autonomous parvoviruses, with Tϭ1 icosahedral symmetry and an overall diameter of ϳ260 Å that encapsulates a singlestranded-DNA (ssDNA) genome of ϳ5,000 bases (9,40,53,62,70). The capsid consists of three overlapping viral proteins (VPs), namely, VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa), at a ratio of 1:1:10, that are generated by alternative splicing and translational initiation during productive infection. VP3 constitutes 90% of the capsid, but all three proteins contain a common C-terminal domain of about 530 amino acids.Recombinant gene transfer vectors based on AAV serotype 2 (AAV2) have proven effective in animal models for the correction of genetic diseases of the eye, brain, muscle, liver, and lung (e.g., see references 18-20, 26, and 58), with human clinical trials now under way for the correction of several diseases. However, numerous studies, including those that use "pseudotyped" capsids, in which AAV vectors are generated usin...

Directed evolution of adeno-associated virus to an infectious respiratory virus

“…It is exceedingly difficult to predict which capsid regions should be reengineered to form novel viral-cell interactions that would be optimal to transduce particular tissues, despite advances in understanding AAV structure and function (37)(38)(39)(40). However, the search for AAV variants with novel properties through a variety of algorithms-such as isolating naturally occurring AAV, site-directed mutants, or (most recently) laboratory-evolved variants-has yielded various chimeric AAV viruses with distinct transduction profiles (17,(41)(42)(43)(44).…”

A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection

“…These AAV serotypes exhibit different infectivities on various tissues. Three-dimensional (3D) structure and mutagenesis studies of several AAV serotypes have shown that the common capsid region displays an 8-stranded (bB-bI) core ␤-barrel motif with loop insertions, which are the main determinants of AAV serotypespecific properties, such as receptor recognition, transduction efficiency and antigenic reactivity (15)(16)(17). The structural information on the capsid forms the basis for genetic engineering of novel AAV vectors.…”

Structural Insights into Adeno-Associated Virus Serotype 5

“…The VP3 topology contains a core eightstranded (␤B to ␤I) ␤-barrel motif and large loop insertions between the ␤-strands which form the surface of the capsid. Conformational variations in these surface loops (VR-I to VR-IX [22]) define the unique surface features for the each of the six representative AAV capsid structures, AAV2, AAV3B, AAV4, AAV6, AAV8, and AAV9, previously determined by X-ray crystallography (21-23, 26, 27, 29).Several wild-type AAV isolates as well as chimeras generated by directed evolution or rational mutagenesis are actively being developed for gene delivery applications (e.g., reviewed in reference 2). Transgenes packaged within the prototype human serotype…”