Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding

The adeno-associated virus type 2 (AAV2) uses heparan sulfate proteoglycan (HSPG) as its primary cellular receptor. In order to identify amino acids within the capsid of AAV2 that contribute to HSPG association, we used biochemical information about heparin and heparin sulfate, AAV serotype protein sequence alignments, and data from previous capsid studies to select residues for mutagenesis. Charged-to-alanine substitution mutagenesis was performed on individual residues and combinations of basic residues for the production and purification of recombinant viruses that contained a green fluorescent protein (GFP) reporter gene cassette. Intact capsids were assayed for their ability to bind to heparin-agarose in vitro, and virions that packaged DNA were assayed for their ability to transduce normally permissive cell lines. We found that mutation of arginine residues at position 585 or 588 eliminated binding to heparin-agarose. Mutation of residues R484, R487, and K532 showed partial binding to heparin-agarose. We observed a general correlation between heparin-agarose binding and infectivity as measured by GFP transduction; however, a subset of mutants that partially bound heparin-agarose (R484A and K532A) were completely noninfectious, suggesting that they had additional blocks to infectivity that were unrelated to heparin binding. Conservative mutation of positions R585 and R588 to lysine slightly reduced heparin-agarose binding and had comparable effects on infectivity. Substitution of AAV2 residues 585 through 590 into a location predicted to be structurally equivalent in AAV5 generated a hybrid virus that bound to heparin-agarose efficiently and was able to package DNA but was noninfectious. Taken together, our results suggest that residues R585 and R588 are primarily responsible for heparin sulfate binding and that mutation of these residues has little effect on other aspects of the viral life cycle. Interactive computer graphics examination of the AAV2 VP3 atomic coordinates revealed that residues which contribute to heparin binding formed a cluster of five basic amino acids that presented toward the icosahedral threefold axis from the surrounding spike protrusion. Three other kinds of mutants were identified. Mutants R459A, H509A, and H526A/K527A bound heparin at levels comparable to that of wild-type virus but were defective for transduction. Another mutant, H358A, was defective for capsid assembly. Finally, an R459A mutant produced significantly lower levels of full capsids, suggesting a packaging defect.     

Identification and characterization of novel adeno-associated virus isolates in ATCC virus stocks

Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for cell binding and transduction, differences were observed in lectin competition, resulting in distinct tropisms in human cancer cell lines.     

Terminal N-linked galactose is the primary receptor for adeno-associated virus 9

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.     

Adeno-associated virus type 2 contains an integrin alpha5beta1 binding domain essential for viral cell entry

Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with alphaV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin alphaVbeta5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin alpha5beta1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind alpha5beta1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a "click-to-fit" mechanism that involves the cooperative binding of heparan sulfate and alpha5beta1 integrin by the AAV2 capsids.     

Membrane-Associated Heparan Sulfate Proteoglycan Is a Receptor for Adeno-Associated Virus Type 2 Virions

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.     

Comparative structural,biophysical, and receptor binding study of true type and wild type AAV2

Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (T_m) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in T_m, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.     

Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency

New viral strains can be evolved to recognize different host glycans through mutagenesis and experimental adaptation. However, such mutants generally harbor amino acid changes that affect viral binding to a single class of carbohydrate receptors. We describe the rational design and synthesis of novel, chimeric adeno-associated virus (AAV) strains that exploit an orthogonal glycan receptor for transduction. A dual glycan-binding AAV strain was first engineered as proof of concept by grafting a galactose (Gal)-binding footprint from AAV serotype 9 onto the heparan sulfate-binding AAV serotype 2. The resulting chimera, AAV2G9, continues to bind heparin affinity columns but interchangeably exploits Gal and heparan sulfate receptors for infection, as evidenced by competitive inhibition assays with lectins, glycans, and parental AAV strains. Although remaining hepatotropic like AAV2, the AAV2G9 chimera mediates rapid onset and higher transgene expression in mice. Similarly, engraftment of the Gal footprint onto the laboratory-derived strain AAV2i8 yielded an enhanced AAV2i8G9 chimera. This new strain remains liver-detargeted like AAV2i8 while selectively transducing muscle tissues at high efficiency, comparable with AAV9. The AAV2i8G9 chimera is a promising vector candidate for targeted gene therapy of cardiac and musculoskeletal diseases. In addition to demonstrating the modularity of glycan receptor footprints on viral capsids, our approach provides design strategies to expand the AAV vector toolkit.     

The threefold protrusions of adeno-associated virus type 8 are involved in cell surface targeting as well as postattachment processing

Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the in vivo gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2 cap sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids (588)QQNTA(592) of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process.     

Structure of adeno-associated virus serotype 8, a gene therapy vector

Adeno-associated viruses (AAVs) are being developed as gene therapy vectors, and their efficacy could be improved by a detailed understanding of their viral capsid structures. AAV serotype 8 (AAV8) shows a significantly greater liver transduction efficiency than those of other serotypes, which has resulted in efforts to develop this virus as a gene therapy vector for hemophilia A and familial hypercholesterolemia. Pseudotyping studies show that the differential tissue tropism and transduction efficiencies exhibited by the AAVs result from differences in their capsid viral protein (VP) amino acids. Towards identifying the structural features underpinning these disparities, we report the crystal structure of the AAV8 viral capsid determined to 2.6-A resolution. The overall topology of its common overlapping VP is similar to that previously reported for the crystal structures of AAV2 and AAV4, with an eight-stranded beta-barrel and long loops between the beta-strands. The most significant structural differences between AAV8 and AAV2 (the best-characterized serotype) are located on the capsid surface at protrusions surrounding the two-, three-, and fivefold axes at residues reported to control transduction efficiency and antibody recognition for AAV2. In addition, a comparison of the AAV8 and AAV2 capsid surface amino acids showed a reduced distribution of basic charge for AAV8 at the mapped AAV2 heparin sulfate receptor binding region, consistent with an observed non-heparin-binding phenotype for AAV8. Thus, this AAV8 structure provides an additional platform for mutagenesis efforts to characterize AAV capsid regions responsible for differential cellular tropism, transduction, and antigenicity for these promising gene therapy vectors.     

Cross-Packaging of a Single Adeno-Associated Virus (AAV) Type 2 Vector Genome into Multiple AAV Serotypes Enables Transduction with Broad Specificity

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 x 10(11) and 9.8 x 10(12)/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.     

Hepatocyte growth factor receptor is a coreceptor for adeno-associated virus type 2 infection

After the first attachment of virus to the cell surface through a primary receptor, efficient entry of virus requires the presence of a coreceptor. For adeno-associated virus type 2 (AAV2) infection, heparan sulfate proteoglycan is supposed as the primary receptor, and alphavbeta5 integrin and FGFR1 are reported to act as coreceptors. In this study, we were able to demonstrate that hepatocyte growth factor receptor, c-Met, is also a coreceptor for AAV2 infection. AAV2-mediated transgene analyses revealed that c-Met expression significantly up-regulated transgene expression without increasing AAV2 cell binding. Moreover, a viral overlay assay elucidated the physical interaction between AAV2 and the beta subunit of c-Met. These data suggest that c-Met plays the role of coreceptor for AAV2 infection by facilitating AAV2 internalization into the cytoplasm.     

Alpha2,3 and alpha2,6 N-linked sialic acids facilitate efficient binding and transduction by adeno-associated virus types 1 and 6

Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either alpha2,3-linked or alpha2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcalpha2-3GalNAcbeta1-4GlcNAc, as well as two glycoproteins with alpha2,3 and alpha2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support alpha2,3 and alpha2,6 sialic acids that are present on N-linked glycoproteins as primary receptors for efficient AAV1 and AAV6 viral infection.     

Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46

NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.     

Adeno-associated virus type 12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate proteoglycan-independent transduction activity

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy. Recent isolations of novel AAV serotypes have led to significant advances by broadening the tropism and increasing the efficiency of gene transfer to the desired target cell. However, a major concern that remains is the strong preexisting immune responses to several vectors. In this paper, we describe the isolation and characterization of AAV12, an AAV serotype with unique biological and immunological properties. In contrast to those of all other reported AAVs, AAV12 cell attachment and transduction do not require cell surface sialic acids or heparan sulfate proteoglycans. Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. The feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications.     

Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation

The early steps of adeno-associated virus (AAV) infection involve attachment to a variety of cell surface receptors (heparan sulfate, integrins, and fibroblast growth factor receptor 1) followed by clathrin-dependent or independent internalization. Here we have studied the subsequent intracellular trafficking of AAV particles from the endosomal compartment to the nucleus. Human cell lines were transduced with a recombinant AAV (rAAV) carrying a reporter gene (luciferase or green fluorescent protein) in the presence of agents that affect trafficking. The effects of bafilomycin A(1), brefeldin A, and MG-132 were measured. These drugs act at the level of endosome acidification, early-to-late endosome transition, and proteasome activity, respectively. We observed that the transducing virions needed to be routed as far as the late endosomal compartment. This behavior was markedly different from that observed with adenovirus particles. Antiproteasome treatments with MG-132 led to a 50-fold enhancement in transduction efficiency. This effect was accompanied by a 10-fold intracellular accumulation of single-stranded DNA AAV genomes, suggesting that the mechanism of transduction enhancement was different from the one mediated by a helper adenovirus, which facilitates the conversion of the rAAV single-stranded DNA genome into its replicative form. MG-132, a drug currently in clinical use, could be of practical use for potentializing rAAV-mediated delivery of therapeutic genes.     

Recombinant Adeno-Associated Virus: Efficient Transduction of the Rat VMH and Clearance from Blood

To promote the efficient and safe application of adeno-associated virus (AAV) vectors as a gene transfer tool in the central nervous system (CNS), transduction efficiency and clearance were studied for serotypes commonly used to transfect distinct areas of the brain. As AAV2 was shown to transduce only small volumes in several brain regions, this study compares the transduction efficiency of three AAV pseudotyped vectors, namely AAV2/1, AAV2/5 and AAV2/8, in the ventromedial nucleus of the hypothalamus (VMH). No difference was found between AAV2/1 and AAV2/5 in transduction efficiency. Both AAV2/1 and AAV2/5 achieved a higher transduction rate than AAV2/8. One hour after virus administration to the brain, no viral particles could be traced in blood, indicating that no or negligible numbers of virions crossed the blood-brain barrier. In order to investigate survival of AAV in blood, clearance was determined following systemic AAV administration. The half-life of AAV2/1, AAV2/2, AAV2/5 and AAV2/8 was calculated by determining virus clearance rates from blood after systemic injection. The half-life of AAV2/2 was 4.2 minutes, which was significantly lower than the half-lives of AAV2/1, AAV2/5 and AAV2/8. With a half-life of more than 11 hours, AAV2/8 particles remained detectable in blood significantly longer than AAV2/5. We conclude that application of AAV in the CNS is relatively safe as no AAV particles are detectable in blood after injection into the brain. With a half-life of 1.67 hours of AAV2/5, a systemic injection with 1×10⁹ genomic copies of AAV would be fully cleared from blood after 2 days.     

Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups

For all adeno-associated virus (AAV) serotypes, 60 monomers of the Vp1, Vp2, and Vp3 structural proteins assemble via an unknown mechanism to form an intact capsid. In an effort to better understand the properties of the capsid monomers and their role in viral entry and infection, we evaluated whether monomers from distinct serotypes can be mixed to form infectious particles with unique phenotypes. This transcapsidation approach consisted of the transfection of pairwise combinations of AAV serotype 1 to 5 helper plasmids to produce mosaic capsid recombinant AAV (rAAV). All ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these mixtures were able to replicate the green fluorescent protein transgene and to produce capsid proteins. A high-titer rAAV was obtained with mixtures that included either serotype 1, 2, or 3, whereas an rAAV of intermediate titer was obtained from serotype 5 mixtures. Only mixtures containing the AAV4 capsid exhibited reduced packaging capacity. The binding profiles of the mixed-virus preparations to either heparin sulfate (HS) or mucin agarose revealed that only AAV3-AAV5 mixtures at the 3:1 ratio exhibited duality in binding. All other mixtures displayed either an abrupt shift or a gradual alteration in the binding profile to the respective ligand upon increase of a capsid component that conferred either HS or mucin binding. The transduction of cell lines was used to further evaluate the phenotypes of these transcapsidated virions. Three transduction profiles were observed: (i) small to no change regardless of ratio, (ii) a gradual increase in transduction consistent with titration of a second capsid component, or (iii) an abrupt increase in transduction (threshold effect) dependent on the specific ratios used. Interestingly, an unexpected synergistic effect in transduction was observed when AAV1 helper constructs were combined with type 2 or type 3 recipient helpers. Further studies determined that at least two components contributed to this observed synergy: (i) heparin-mediated binding from AAV2 and (ii) an unidentified enhancement activity from AAV1 structural proteins. Using this procedure of mixing different AAV helper plasmids to generate "cross-dressed" AAV virions, we propose an additional means of classifying new AAV serotypes into subgroups based on functional approaches to analyze AAV capsid assembly, receptor-mediated binding, and virus trafficking. Exploitation of this approach in generating custom-designed AAV vectors should be of significant value to the field of gene therapy.     

Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells

Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated.     

A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.     

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8(+) T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.