The structure of the adeno-associated virus (AAV-2) has been determined to 3-Å resolution by x-ray crystallography. AAV is being developed as a vector for gene therapy to treat diseases including hemophilia, cancer, and cystic fibrosis. As in the distantly related autonomous parvoviruses, the capsid protein has a β-barrel fold, but long loops between the β-strands share little structural homology with other parvoviruses, leading to unique surface features. Most prominent are groups of threefold-related peaks, each an intimate association of loops from two neighboring subunits. Mutations affecting cell entry and receptor binding are clustered near the positively charged side of each peak, implicating the region in attachment to the cellular receptor, heparan sulfate proteoglycan. Amino acids involved in antibody binding are in the same general vicinity. The structure will guide rational engineering of vector capsids to tailor cellular targeting and to avoid immediate neutralization by an immune system sensitized by prior exposure to AAV.

An adeno-associated virus (AAV) receptor protein essential for AAV2 entry into cells is identified; AAV receptor binds directly to the virus, and its ablation renders a diverse range of mammalian cell types and mice resistant to infection by AAV of multiple serotypes. The recent revival of interest in gene therapy has been fueled by the availability of safer and more effective viral gene delivery methods, most notably adeno-associated virus (AAV) vectors. Jan Carette and colleagues now identify a protein that is essential for AAV entry into cells, subsequent to cell attachment. This protein, which they call AAVR, rapidly traffics from the plasma membrane to the trans-Golgi network. The authors show that the virus directly binds to AAVR and that genetic ablation of AAVR renders a diverse range of mammalian cell types and mice resistant to AAV infection. Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity1. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe3. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration1,4, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr−/− (also known as Au040320−/− and Kiaa0319l−/−) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

The three-dimensional atomic structure of a single-stranded DNA virus has been determined. Infectious virions of canine parvovirus contain 60 protein subunits that are predominantly VP-2. The central structural motif of VP-2 has the same topology (an eight-stranded antiparallel β barrel) as has been found in many other icosahedral viruses but represents only about one-third of the capsid protein. There is a 22 angstrom (Å) long protrusion on the threefold axes, a 15 Å deep canyon circulating about each of the five cylindrical structures at the fivefold axes, and a 15 Å deep depression at the twofold axes. By analogy with rhinoviruses, the canyon may be the site of receptor attachment. Residues related to the antigenic properties of the virus are found on the threefold protrusions. Some of the amino termini of VP-2 run to the exterior in full but not empty virions, which is consistent with the observation that some VP-2 polypeptides in full particles can be cleaved by trypsin. Eleven nucleotides are seen in each of 60 symmetry-related pockets on the interior surface of the capsid and together account for 13 percent of the genome.

Arginine kinase belongs to the family of enzymes, including creatine kinase, that catalyze the buffering of ATP in cells with fluctuating energy requirements and that has been a paradigm for classical enzymological studies. The 1.86-Å resolution structure of its transition-state analog complex, reported here, reveals its active site and offers direct evidence for the importance of precise substrate alignment in the catalysis of bimolecular reactions, in contrast to the unimolecular reactions studied previously. In the transition-state analog complex studied here, a nitrate mimics the planar γ-phosphoryl during associative in-line transfer between ATP and arginine. The active site is unperturbed, and the reactants are not constrained covalently as in a bisubstrate complex, so it is possible to measure how precisely they are pre-aligned by the enzyme. Alignment is exquisite. Entropic effects may contribute to catalysis, but the lone-pair orbitals are also aligned close enough to their optimal trajectories for orbital steering to be a factor during nucleophilic attack. The structure suggests that polarization, strain toward the transition state, and acid-base catalysis also contribute, but, in contrast to unimolecular enzyme reactions, their role appears to be secondary to substrate alignment in this bimolecular reaction.