Structural Insights into Adeno-Associated Virus Serotype 5

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Bovine parvovirus (BPV), the causative agent of respiratory and gastrointestinal disease in cows, is the type member of the Bocaparvovirus genus of the Parvoviridae family. Toward efforts to obtain a template for the development of vaccines and small-molecule inhibitors for this pathogen, the structure of the BPV capsid, assembled from the major capsid viral protein 2 (VP2), was determined using X-ray crystallography as well as cryo-electron microscopy and three-dimensional image reconstruction (cryoreconstruction) to 3.2-and 8.8-Å resolutions, respectively. The VP2 region ordered in the crystal structure, from residues 39 to 536, conserves the parvoviral eight-stranded jellyroll motif and an ␣A helix. The BPV capsid displays common parvovirus features: a channel at and depressions surrounding the 5-fold axes and protrusions surrounding the 3-fold axes. However, rather than a depression centered at the 2-fold axes, a raised surface loop divides this feature in BPV. Additional observed density in the capsid interior in the cryo-reconstructed map, compared to the crystal structure, is interpreted as 10 additional N-terminal residues, residues 29 to 38, that radially extend the channel under the 5-fold axis, as observed for human bocavirus 1 (HBoV1). Surface loops of various lengths and conformations extend from the core jellyroll motif of VP2. These loops confer the unique surface topology of the BPV capsid, making it strikingly different from HBoV1 as well as the type members of other Parvovirinae genera for which structures have been determined. For the type members, regions structurally analogous to those decorating the BPV capsid surface serve as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity. IMPORTANCEBovine parvovirus (BPV), identified in the 1960s in diarrheic calves, is the type member of the Bocaparvovirus genus of the nonenveloped, single-stranded DNA (ssDNA) Parvoviridae family. The recent isolation of human bocaparvoviruses from children with severe respiratory and gastrointestinal infections has generated interest in understanding the life cycle and pathogenesis of these emerging viruses. We have determined the high-resolution structure of the BPV capsid assembled from its predominant capsid protein VP2, known to be involved in a myriad of functions during host cell entry, pathogenesis, and antigenicity for other members of the Parvovirinae. Our results show the conservation of the core secondary structural elements and the location of the N-terminal residues for the known bocaparvovirus capsid structures. However, surface loops with high variability in sequence and conformation give BPV a unique capsid surface topology. Similar analogous regions in other Parvovirinae type members are important as determinants of receptor recognition, tissue and host tropism, pathogenicity, and antigenicity. B ovine parvovirus (BPV), the type member of the Bocaparvovirus genus of the Parvoviridae family, was discovered in 1961 in the gastrointestinal tract of di...

Section: Resultsmentioning

confidence: 98%

Structure of an Enteric Pathogen, Bovine Parvovirus

Kailasan

Halder

Gurda

et al. 2015

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“…In this region, AAV5 differs from the other serotypes by the lack of observable cleavage (Table 4). AAV5 is also different with respect to the 3-fold protrusion, which is less pronounced than in the other serotypes (45,104), and it is predicted to have a strong propensity for disorder across VR5 to -8 based on PONDR (74). Data from mutagenesis and structural studies localize the known primary receptor binding sites to the region surrounding the 3-fold axis (15-17, 19, 88, 97).…”

Section: Discussionmentioning

confidence: 99%

Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Rayaprolu

Kruse

Kant

et al. 2013

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128

108

bIcosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.

“…This is isomorphous with the published AAV5 VLP structure (PDB accession no. 3NTT) (19). Thus, a difference map, F o -F c (where F o and F c are the observed and calculated structure factor amplitudes, respectively), was calculated to localize the potential binding site for SIA using the CNS program (20).…”

Section: Cell Cultures African Green Monkey Kidney Cos Cells (The Ammentioning

confidence: 99%

Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

Afione

DiMattia

Halder

et al. 2015

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As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the ␤HI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus. IMPORTANCEDespite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild-type virus.