Members of the Parvoviridae utilize glycan receptors for cellular attachment and subsequent interactions determine transduction efficiency or pathogenic outcome. This review focuses on the identity of the glycan receptors utilized, their capsid binding footprints, and a discussion of the overlap of these sites with tropism, transduction, and pathogenicity determinants. Despite high sequence diversity between the different genera, most parvoviruses bind to negatively charged glycans, such as sialic acid and heparan sulfate, abundant on cell surface membranes. The capsid structure of these viruses exhibit high structural homology enabling common regions to be utilized for glycan binding and at the same time the sequence diversity at the common footprints allows for binding of different glycans or differential binding of the same glycan.

Background: Recognition of human milk glycans (HMGs) by lectins, antibodies, and pathogens is poorly understood. Results: Microarrays of isolated HMGs exhibited specific binding to proteins and pathogens. Conclusion: HMG microarray interrogation and novel metadata-assisted glycan sequencing provide a functional glycomics approach to discovering HMG function. Significance: HMGs represent a potential "liquid innate immune system" that is specifically recognized by antibodies and pathogens.

Parvoviruses package a ssDNA genome. Both nonpathogenic and pathogenic members exist, including those that cause fetal infections, encompassing the entire spectrum of virus phenotypes. Their small genomes and simple coding strategy has enabled functional annotation of many steps in the infectious life cycle. They assemble a multifunctional capsid responsible for cell recognition and the transport of the packaged genome to the nucleus for replication and progeny virus production. It is also the target of the host immune response. Understanding how the capsid structure relates to the function of parvoviruses provides a platform for recombinant engineering of viral gene delivery vectors for the treatment of clinical diseases, and is fundamental for dissecting the viral determinants of pathogenicity. This review focuses on our current understanding of parvovirus capsid structure and function with respect to the infectious life cycle.

The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wildtype AAV6. Furthermore, three of the AAV1 SIA binding site mutants-S472R, V473D, and N500E-escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface "hot spot" dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering. IMPORTANCEThe adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and the detrimental effects of preexisting host antibodies. This study aimed to identify key capsid regions which can be engineered to overcome these limitations. A sialic glycan receptor recognition pocket was identified in AAV1 and its closely related AAV6, using X-ray crystallography. The site was confirmed by mutagenesis followed by cell binding and transduction assays. Significantly, residues controlling gene expression efficiency, as well as antibody escape variants, were also identified. This study thus provides, at the amino acid level, information for rational structural engineering of AAV vectors with improved therapeutic efficacy.

The adeno-associated viruses (AAVs) display differential cell binding, transduction, and antigenic characteristics specified by their capsid viral protein (VP) composition. Toward structure-function annotation, the crystal structure of AAV5, one of the most sequence diverse AAV serotypes, was determined to 3.45-Å resolution. The AAV5 VP and capsid conserve topological features previously described for other AAVs but uniquely differ in the surface-exposed HI loop between ␤H and ␤I of the core ␤-barrel motif and have pronounced conformational differences in two of the AAV surface variable regions (VRs), VR-IV and VR-VII. The HI loop is structurally conserved in other AAVs despite amino acid differences but is smaller in AAV5 due to an amino acid deletion. This HI loop is adjacent to VR-VII, which is largest in AAV5. The VR-IV, which forms the larger outermost finger-like loop contributing to the protrusions surrounding the icosahedral 3-fold axes of the AAVs, is shorter in AAV5, creating a smoother capsid surface topology. The HI loop plays a role in AAV capsid assembly and genome packaging, and VR-IV and VR-VII are associated with transduction and antigenic differences, respectively, between the AAVs. A comparison of interior capsid surface charge and volume of AAV5 to AAV2 and AAV4 showed a higher propensity of acidic residues but similar volumes, consistent with comparable DNA packaging capacities. This structure provided a three-dimensional (3D) template for functional annotation of the AAV5 capsid with respect to regions that confer assembly efficiency, dictate cellular transduction phenotypes, and control antigenicity. R ecombinant adeno-associated viruses (rAAVs) are promising viral vectors for gene delivery applications (1, 2). These viruses belong to the single-stranded DNA (ssDNA)-packaging Parvoviridae and genus Dependovirus. Hundreds of AAV genotypes have been sequenced from several mammalian species, and to date 12 serotypes (AAV1 to AAV12) have been defined for the human and nonhuman primate isolates (3-15). The 12 serotypes are classified into eight genetic groups, clades A to F and clonal isolates AAV4 and AAV5, based on antigenic reactivity and sequence similarity (15). The groups are represented by AAV1 to AAV9, with AAV1 and AAV6 belonging to the same clade A because of their high sequence similarity and antigenic cross-reactivity. The representative members share ϳ55 to 99% sequence identity, with AAV4 and AAV5 being the most divergent from each other and from the other members.The linear ssDNA AAV genome, ϳ4.7 kb in length, contains two genes: cap, which encodes the capsid viral proteins (VPs; VP1, ϳ87 kDa; VP2, ϳ73 kDa; VP3, ϳ62 kDa) and rep, which encodes the replication proteins necessary for genome replication and genome packaging. Inverted terminal repeats (ITRs) at the end of the AAV genome, 145 bp in length, are the only essential active sequence required to function as (i) the origin for DNA replication, (ii) the packaging signal, and (iii) integration sites (16)(17)(18)(19). Recombina...

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...

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.

bThe structure of single-stranded DNA (ssDNA) packaging H-1 parvovirus (H-1PV), which is being developed as an antitumor gene delivery vector, has been determined for wild-type (wt) virions and noninfectious (empty) capsids to 2.7-and 3.2-Å resolution, respectively, using X-ray crystallography. The capsid viral protein (VP) structure consists of an ␣-helix and an eightstranded anti-parallel ␤-barrel with large loop regions between the strands. The ␤-barrel and loops form the capsid core and surface, respectively. In the wt structure, 600 nucleotides are ordered in an interior DNA binding pocket of the capsid. This accounts for ϳ12% of the H-1PV genome. The wt structure is identical to the empty capsid structure, except for side chain conformation variations at the nucleotide binding pocket. Comparison of the H-1PV nucleotides to those observed in canine parvovirus and minute virus of mice, two members of the genus Parvovirus, showed both similarity in structure and analogous interactions. This observation suggests a functional role, such as in capsid stability and/or ssDNA genome recognition for encapsulation. The VP structure differs from those of other parvoviruses in surface loop regions that control receptor binding, tissue tropism, pathogenicity, and antibody recognition, including VP sequences reported to determine tumor cell tropism for oncotropic rodent parvoviruses. These structures of H-1PV provide insight into structural features that dictate capsid stabilization following genome packaging and three-dimensional information applicable for rational design of tumor-targeted recombinant gene delivery vectors. H -1 parvovirus (H-1PV) is a member of the rodent subgroup of the Parvovirus genus of the single-stranded DNA (ssDNA) Parvoviridae (1). H-1PV was first isolated from rats transplanted with HEP-1, a human liver adenocarcinoma cell line (2), and also from aborted human fetuses (3). Recombinant vectors based on H-1PV and a number of other rodent parvoviruses, including minute virus of mice (MVM) and LuIII, are promising candidates for antitumor delivery vectors, particularly for cytoreductive and immunogene therapy approaches (reviewed in references 4 and 5). The inherent oncotropism of these autonomous parvoviruses is based on their dependence on cellular proliferation factors expressed during the S phase and the differentiated state of the host cell (6, 7). The rodent parvoviruses display oncopreferential cytotoxic activity in vitro and also possess an oncosuppressive potential, inhibiting the formation of spontaneous and chemical or virus-induced tumors in vitro and in vivo (8-13). These viruses can also persistently infect their natural hosts, do not integrate their genome into cellular chromosomes, and are not associated with human disease (reviewed in reference 4).Recombinant rodent parvovirus vectors targeted for tumor therapy utilize a double-edged strategy that takes advantage of their inherent oncotropism and selective cytotoxicity plus their ability to deliver therapeutic genes that code for ...