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1.
Govindasamy L Padron E McKenna R Muzyczka N Kaludov N Chiorini JA Agbandje-McKenna M 《Journal of virology》2006,80(23):11556-11570
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 beta strands (betaB to betaI) and helical (alphaA) 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. 相似文献
2.
Walters RW Agbandje-McKenna M Bowman VD Moninger TO Olson NH Seiler M Chiorini JA Baker TS Zabner J 《Journal of virology》2004,78(7):3361-3371
Adeno-associated virus serotype 5 (AAV5) requires sialic acid on host cells to bind and infect. Other parvoviruses, including Aleutian mink disease parvovirus (ADV), canine parvovirus (CPV), minute virus of mice, and bovine parvovirus, also bind sialic acid. Hence, structural homology may explain this functional homology. The amino acids required for CPV sialic acid binding map to a site at the icosahedral twofold axes of the capsid. In contrast to AAV5, AAV2 does not bind sialic acid, but rather binds heparan sulfate proteoglycans at its threefold axes of symmetry. To explore the structure-function relationships among parvoviruses with respect to cell receptor attachment, we determined the structure of AAV5 by cryo-electron microscopy (cryo-EM) and image reconstruction at a resolution of 16 A. Surface features common to some parvoviruses, namely depressions encircling the fivefold axes and protrusions at or surrounding the threefold axes, are preserved in the AAV5 capsid. However, even though there were some similarities, a comparison of the AAV5 structure with those of ADV and CPV failed to reveal a feature which could account for the sialic acid binding phenotype common to all three viruses. In contrast, the overall surface topologies of AAV5 and AAV2 are similar. A pseudo-atomic model generated for AAV5 based on the crystal structure of AAV2 and constrained by the AAV5 cryo-EM envelope revealed differences only in surface loop regions. Surprisingly, the surface topologies of AAV5 and AAV2 are remarkably similar to that of ADV despite only exhibiting approximately 20% identity in amino acid sequences. Thus, capsid surface features are shared among parvoviruses and may not be unique to their replication phenotypes, i.e., whether they require a helper or are autonomous. Furthermore, specific surface features alone do not explain the variability in carbohydrate requirements for host cell receptor interactions among parvoviruses. 相似文献
3.
Three-Dimensional Structure of Aleutian Mink Disease Parvovirus: Implications for Disease Pathogenicity 总被引:14,自引:0,他引:14
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Robert McKenna Norman H. Olson Paul R. Chipman Timothy S. Baker Tim F. Booth Jesper Christensen Bent Aasted James M. Fox Marshall E. Bloom James B. Wolfinbarger Mavis Agbandje-McKenna 《Journal of virology》1999,73(8):6882-6891
The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples. 相似文献
4.
Nam HJ Lane MD Padron E Gurda B McKenna R Kohlbrenner E Aslanidi G Byrne B Muzyczka N Zolotukhin S Agbandje-McKenna M 《Journal of virology》2007,81(22):12260-12271
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. 相似文献
5.
Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes
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Wu Z Asokan A Grieger JC Govindasamy L Agbandje-McKenna M Samulski RJ 《Journal of virology》2006,80(22):11393-11397
Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors. 相似文献
6.
Lakshmanan Govindasamy Michael A. DiMattia Brittney L. Gurda Sujata Halder Robert McKenna John A. Chiorini Nicholas Muzyczka Sergei Zolotukhin Mavis Agbandje-McKenna 《Journal of virology》2013,87(20):11187-11199
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 βH and βI of the core β-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. 相似文献
7.
Brittney L. Gurda Michael A. DiMattia Edward B. Miller Antonette Bennett Robert McKenna Wendy S. Weichert Christian D. Nelson Wei-jun Chen Nicholas Muzyczka Norman H. Olson Robert S. Sinkovits John A. Chiorini Sergei Zolotutkhin Olga G. Kozyreva R. Jude Samulski Timothy S. Baker Colin R. Parrish Mavis Agbandje-McKenna 《Journal of virology》2013,87(16):9111-9124
Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies. 相似文献
8.
Characterization of the capsid protein glycosylation of adeno-associated virus type 2 by high-resolution mass spectrometry
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Murray S Nilsson CL Hare JT Emmett MR Korostelev A Ongley H Marshall AG Chapman MS 《Journal of virology》2006,80(12):6171-6176
Adeno-associated virus type 2 (AAV-2) capsid proteins have eight sequence motifs that are potential sites for O- or N-linked glycosylation. Three are in prominent surface locations, close to the sites of cellular receptor attachment and to neutralizing epitopes on or near protrusions surrounding the three-fold axes, raising the possibility that AAV-2 might use glycosylation as a means of immune escape or for preventing reattachment on release of progeny virus. Peptide mapping and structural analysis by Fourier transform ion cyclotron resonance mass spectrometry demonstrates, however, no glycosylation of the capsid protein for virus prepared in cultured HeLa cells. 相似文献
9.
Bell CL Gurda BL Van Vliet K Agbandje-McKenna M Wilson JM 《Journal of virology》2012,86(13):7326-7333
Adeno-associated virus serotype 9 (AAV9) vectors show promise for gene therapy of a variety of diseases due to their ability to transduce multiple tissues, including heart, skeletal muscle, and the alveolar epithelium of the lung. In addition, AAV9 is unique compared to other AAV serotypes in that it is capable of surpassing the blood-brain barrier and transducing neurons in the brain and spinal cord. It has recently been shown that AAV9 uses galactose as a receptor to transduce many different cell types in vitro, as well as cells of the mouse airway in vivo. In this study, we sought to identify the specific amino acids of the AAV9 capsid necessary for binding to galactose. By site-directed mutagenesis and cell binding assays, plus computational ligand docking studies, we discovered five amino acids, including N470, D271, N272, Y446, and W503, which are required for galactose binding that form a pocket at the base of the protrusions around the icosahedral 3-fold axes of symmetry. The importance of these amino acids for tissue tropism was also confirmed by in vivo studies in the mouse lung. Identifying the interactions necessary for AAV9 binding to galactose may lead to advances in vector engineering. 相似文献
10.
Adeno-associated virus (AAV) is frequently used as a vector for gene therapy. The viral capsid consists of three structural proteins (VP1, VP2, and VP3) that have a common C-terminal core (VP3), with N-terminal extensions of increasing length in VP2 and VP1. The capsid encloses a single-stranded genome of up to 4.7 kb, which is packaged into empty capsids. The N-terminal extension of VP1 carries a phospholipase domain that becomes accessible during infection in the endosomal pathway. We have used cryo-electron microscopy and image reconstruction to determine subnanometer-resolution structures of recombinant AAV1 that has packaged different amounts of a 3. 6-kb recombinant genome. The maps show that the AAV1 capsid undergoes continuous conformational changes upon packaging of the genome. The rearrangements occur at the inner capsid surface and lead to constrictions of the pores at the 5-fold symmetry axes and to subtle movements of the β-sheet regions of the capsid proteins. In fully packaged particles, the genome forms stem-like features that contact the inner capsid surface at the 3-fold symmetry axes. We think that the reorganization of the inner surface has an impact on the viral life cycle during infection, preparing the externalization of phospholipase domains through the pores at the 5-fold symmetry axes and possibly genome release. 相似文献
11.
Kern A Schmidt K Leder C Müller OJ Wobus CE Bettinger K Von der Lieth CW King JA Kleinschmidt JA 《Journal of virology》2003,77(20):11072-11081
Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary. 相似文献
12.
Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding 总被引:9,自引:0,他引:9
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Opie SR Warrington KH Agbandje-McKenna M Zolotukhin S Muzyczka N 《Journal of virology》2003,77(12):6995-7006
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. 相似文献
13.
Gurda BL Raupp C Popa-Wagner R Naumer M Olson NH Ng R McKenna R Baker TS Kleinschmidt JA Agbandje-McKenna M 《Journal of virology》2012,86(15):7739-7751
Adeno-associated viruses (AAVs) are small single-stranded DNA viruses that can package and deliver nongenomic DNA for therapeutic gene delivery. AAV8, a liver-tropic vector, has shown great promise for the treatment of hemophilia A and B. However, as with other AAV vectors, host anti-capsid immune responses are a deterrent to therapeutic success. To characterize the antigenic structure of this vector, cryo-electron microscopy and image reconstruction (cryo-reconstruction) combined with molecular genetics, biochemistry, and in vivo approaches were used to define an antigenic epitope on the AAV8 capsid surface for a neutralizing monoclonal antibody, ADK8. Docking of the crystal structures of AAV8 and a generic Fab into the cryo-reconstruction for the AAV8-ADK8 complex identified a footprint on the prominent protrusions that flank the 3-fold axes of the icosahedrally symmetric capsid. Mutagenesis and cell-binding studies, along with in vitro and in vivo transduction assays, showed that the major ADK8 epitope is formed by an AAV variable region, VRVIII (amino acids 586 to 591 [AAV8 VP1 numbering]), which lies on the surface of the protrusions facing the 3-fold axis. This region plays a role in AAV2 and AAV8 cellular transduction. Coincidently, cell binding and trafficking assays indicate that ADK8 affects a postentry step required for successful virus trafficking to the nucleus, suggesting a probable mechanism of neutralization. This structure-directed strategy for characterizing the antigenic regions of AAVs can thus generate useful information to help re-engineer vectors that escape host neutralization and are hence more efficacious. 相似文献
14.
Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups
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Rabinowitz JE Bowles DE Faust SM Ledford JG Cunningham SE Samulski RJ 《Journal of virology》2004,78(9):4421-4432
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. 相似文献
15.
The 37/67-kilodalton laminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9 总被引:7,自引:0,他引:7
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Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy. 相似文献
16.
Michelfelder S Varadi K Raupp C Hunger A Körbelin J Pahrmann C Schrepfer S Müller OJ Kleinschmidt JA Trepel M 《PloS one》2011,6(8):e23101
Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes. 相似文献
17.
Chengwen Li Matt Hirsch Nina DiPrimio Aravind Asokan Kevin Goudy Roland Tisch R. Jude Samulski 《Journal of virology》2009,83(13):6817-6824
A recent clinical trial in patients with hemophilia B has suggested that adeno-associated virus (AAV) capsid-specific cytotoxic T lymphocytes (CTLs) eliminated AAV-transduced hepatocytes and resulted in therapeutic failure. AAV capsids elicit a CTL response in animal models; however, these capsid-specific CTLs fail to kill AAV-transduced target cells in mice. To better model the human clinical trial data in mice, we introduced an immunodominant epitope derived from ovalbumin (OVA; SIINFEKL) into the AAV capsid and tested CTL-mediated killing of AAV2-transduced target tissues in vivo. Initially, in vitro experiments demonstrated both classical class I and cross-presentation of the OVA antigen, following endogenous expression or AAV2-OVA vector transduction, respectively. Furthermore, an OVA-specific CTL response was elicited after muscular or systemic injection of the AAV2-OVA vector. Finally, CTL reactivity was enhanced in mice with established SIINFEKL-specific immunity after AAV2-OVA/α1 anti-trypsin (AAT) administration. Most importantly, these OVA-specific CTLs decreased AAT expression in mice treated with AAV2-OVA/AAT vector that followed a time course mimicking uncoating kinetics of AAV2 transduction in OVA-immunized mice. These results demonstrate that AAV capsid-derived antigens elicit CD8+ CTL reactivity, and these CTLs eliminated AAV-transduced target cells in mice. Notably, this model system can be exploited to study the kinetics of capsid presentation from different serotypes of AAV and permit the design of novel strategies to block CTL-mediated killing of AAV-transduced cells.Adeno-associated virus (AAV) is a single-stranded DNA parvovirus. Its replication relies on coinfection of a helper virus such as adenovirus or herpesvirus. In the absence of a helper virus, AAV establishes latency to integrate into the AAVS1 site of host chromosome 19 (11). The genome of AAV is ∼4.7 kb and contains two open reading frames encoding replication proteins and structural capsid proteins (21). The capsid proteins (VP) are composed of VP1, VP2, and VP3. The VP3 protein is the major structural component and constitutes nearly 80% of the virion shell with an overall ratio of 1:1:8 for VP1, VP2, VP3, respectively. While VP2 is thought to be nonessential for AAV transduction (30), the VP1 subunit contains a phospholipase A2 domain required for infectivity (9). Recombinant AAV (rAAV) vectors require only the 145-bp terminal repeats of the AAV genome in cis and all other viral factors supplied in trans for production (18). rAAV vectors have rapidly gained popularity in gene therapy applications and have proven effective in preclinical studies/clinical trials for a number of diseases (20, 31, 33).AAV vectors mount a potent humoral immune response against capsid in animals and human. However, AAV vectors only contain the therapeutic gene flanked by two 145-bp AAV terminal repeats devoid of any AAV genes(23). In addition, AAV initiates long-term stable therapeutic gene expression in animal models (3-5, 17, 31). Based on these observations AAV has been thought to be relatively nonimmunogenic regarding the induction of cytotoxic T lymphocytes (CTLs) specific for capsid proteins. In spite of all of these observations, the recent clinical trial for hemophilia B (F9) gene therapy has otherwise suggested that AAV2 capsid initiates cell-mediated immunity that eliminates the AAV2 encoding F9 (AAV2/F9) vector transduced liver cells (15). Against this backdrop, numerous attempts to replicate aforementioned observations in animal models have been made. Preliminary results from these studies support direct presentation and cross-presentation of the AAV2 capsid in animal models (6, 12, 13, 22, 29). However, capsid-specific CTLs did not eliminate AAV2-transduced target cells in mice (12, 13, 29), inconsistent with observations made in a clinical trial for hemophilia B with AAV2/F9 gene therapy. A potential explanation for this discrepancy is the weak immunogenicity of the AAV2 capsid in mice. Accordingly, we hypothesized that incorporation of a peptide epitope into the AAV2 capsid would increase immunogenicity of the rAAV and therefore could be exploited to mimic events ongoing in humans and study approaches to block capsid-specific CTL reactivity in mice.We chose to introduce the MHC-H2Kb-restricted SIINFEKL peptide derived from ovalbumin (OVA) into AAV2 capsid. Integration of the OVA epitope into AAV capsids elicited a specific CTL response. Most importantly, after administration of genetically engineered AAV2 vectors into OVA peptide-immunized mice, OVA-specific CTL reactivity was further enhanced, thereby limiting transgene expression in vivo. The modified vector described herein is a potentially valuable tool for future studies focused on developing strategies to evade capsid-specific CTL-mediated elimination of AAV-transduced target cells in animal models. 相似文献
18.
Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection
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Wobus CE Hügle-Dörr B Girod A Petersen G Hallek M Kleinschmidt JA 《Journal of virology》2000,74(19):9281-9293
The previously characterized monoclonal antibodies (MAbs) A1, A69, B1, and A20 are directed against assembled or nonassembled adeno-associated virus type 2 (AAV-2) capsid proteins (A. Wistuba, A. Kern, S. Weger, D. Grimm, and J. A. Kleinschmidt, J. Virol. 71:1341-1352, 1997). Here we describe the linear epitopes of A1, A69, and B1 which reside in VP1, VP2, and VP3, respectively, using gene fragment phage display library, peptide scan, and peptide competition experiments. In addition, MAbs A20, C24-B, C37-B, and D3 directed against conformational epitopes on AAV-2 capsids were characterized. Epitope sequences on the capsid surface were identified by enzyme-linked immunoabsorbent assay using AAV-2 mutants and AAV serotypes, peptide scan, and peptide competition experiments. A20 neutralizes infection following receptor attachment by binding an epitope formed during AAV-2 capsid assembly. The newly isolated antibodies C24-B and C37-B inhibit AAV-2 binding to cells, probably by recognizing a loop region involved in binding of AAV-2 to the cellular receptor. In contrast, binding of D3 to a loop near the predicted threefold spike does not neutralize AAV-2 infection. The identified antigenic regions on the AAV-2 capsid surface are discussed with respect to their possible roles in different steps of the viral life cycle. 相似文献
19.
Robert Ng Lakshmanan Govindasamy Brittney L. Gurda Robert McKenna Olga G. Kozyreva R. Jude Samulski Kristin N. Parent Timothy S. Baker Mavis Agbandje-McKenna 《Journal of virology》2010,84(24):12945-12957
The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization.Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA (ssDNA) parvoviruses that belong to the Dependovirus genus and require helper viruses, such as Adenovirus or Herpesvirus, for lytic infection (4, 8, 22, 67). These viruses package a genome of ∼4.7 kb inside an icosahedral capsid (∼260 Å in diameter) with a triangulation number equal to 1 assembled from a total of 60 copies of their overlapping capsid viral protein (VP) 1 (VP1), VP2, and VP3 in a predicted ratio of 1:1:8/10 (10). The VPs are encoded from a cap open reading frame (ORF). VP3 is 61 kDa and constitutes 90% of the capsid''s protein composition. The less abundant VPs, VP1 (87 kDa) and VP2 (73 kDa), share the same C-terminal amino acid sequence with VP3 but have additional N-terminal sequences. A rep ORF codes for four overlapping proteins required for replication and DNA packaging.To date, more than 100 AAV isolates have been identified (21). Among the human and nonhuman primate AAVs isolated, 12 serotypes (AAV serotype 1 [AAV1] to AAV12) have been described and are classified into six phylogenetic clades on the basis of their VP sequences and antigenic reactivities, with AAV4 and AAV5 considered to be clonal isolates (21). AAV1 and AAV6, which represent clade A, differ by only 6 out of 736 VP1 amino acids (5 amino acids within VP3) and are antigenically cross-reactive. Other clade representatives include AAV2 (clade B), AAV2-AAV3 hybrid (clade C), AAV7 (clade D), AAV8 (clade E), and AAV9 (clade F) (21).The AAVs are under development as clinical gene delivery vectors (e.g., see references 5, 9, 12, 13, 24, 25, 53, and 61), with AAV2, the prototype member of the genus, being the most extensively studied serotype for this application. AAV2 has been successfully used to treat several disorders, but its broad tissue tropism makes it less effective for tissue-specific applications and the prevalence of preexisting neutralizing antibodies in the human population (11, 43) limits its utilization, especially when readministration is required to achieve a therapeutic outcome. Efforts have thus focused on characterizing the capsid-associated tissue tropism and transduction properties conferred by the capsid of representative serotypes of other clades (21). Outcomes of these studies include the observation that AAV1 and AAV6, for example, transduce liver, muscle, and airway epithelial cells more efficiently (e.g., up to 200-fold) than AAV2 (27, 28, 30). In addition, the six residues (Table (Table1)1) that differ between the VPs of AAV1 and AAV6 (a natural recombinant of AAV1 and AAV2 [56]) confer functional disparity between these two viruses. For example, AAV6 shows ∼3-fold higher lung cell epithelium transduction than AAV1 (27), and AAV1 and AAV6 bind terminally sialylated proteoglycans as their primary receptor, whereas AAV6 additionally binds to heparan sulfate (HS) proteoglycans with moderate affinity (70, 71). Therefore, a comparison of the AAV1 and AAV6 serotypes and, in particular, their capsid structures can help pinpoint the capsid regions that confer differences in cellular recognition and tissue transduction.
Open in a separate windowaMutant residues in boldface have an AAV6 parental original; those underlined have an AAV1 parental origin.bS, sialic acid; HS, heparan sulfate; HS+, HS positive.cThe sialic acid binding phenotypes of these mutants were not discussed in the respective publications but are assumed to be still present.dThe glycan targets for these mutants were not discussed in this publication; thus, the phenotypes indicated are assumed.eThis mutant is sialic acid independent (S-ind) for cellular transduction.The structures of AAV1 to AAV5 and AAV8 have been determined by X-ray crystallography and/or cryo-electron microscopy and image reconstruction (cryo-EM) (23, 36, 47, 52, 66, 73; unpublished data), and preliminary characterization of crystals has also been reported for AAV1, AAV5, AAV7, and AAV9 (15, 45, 46, 55). The capsid VP structures contain a conserved eight-stranded (βB to βI) β-barrel core and large loop regions between the strands that form the capsid surface. The capsid surface is characterized by depressions at the icosahedral 2-fold axes of symmetry, finger-like projections surrounding the 3-fold axes, and canyon-like depressions surrounding the 5-fold axes. A total of nine variable regions (VRs; VRI to VRIX) were defined when the two most disparate structures, AAV2 and AAV4, were compared (23). The VRs contain amino acids that contribute to slight differences in surface topologies and distinct functional phenotypes, such as in receptor binding, transduction efficiency, and antigenic reactivity (10, 23, 37, 47).The structure of virus-like particles (VLPs) of AAV6, produced in a baculovirus/Sf9 insect cell expression system, has been determined by two highly complementary approaches, cryo-EM and X-ray crystallography. The AAV6 VP structure contains the general features already described for the AAVs and has conformational differences in the VRs compared to the VRs of other AAVs. The 9.7-Å-resolution cryoreconstructed structure enabled the localization of the C-α positions of five of the six amino acids that differ between highly homologous AAV6 and AAV1 but did not provide information on the positions of the side chains or their orientations. The X-ray crystal structure determined to 3.0-Å resolution enabled us to precisely map the atomic positions of these five residues at or close to the icosahedral 3-fold axes of the capsid. Reported mutagenesis and biochemical studies had functionally annotated the six residues differing between AAV1 and AAV6 with respect to their roles in receptor attachment and differential cellular transduction. Their disposition identifies the 3-fold capsid region as playing essential roles in AAV infection. 相似文献
TABLE 1.
Amino acid differences between AAV1 and AAV6 and their reported mutantsAAV | Amino acid at positiona: | Glycan targetb | Reference | ||||||
---|---|---|---|---|---|---|---|---|---|
129 | 418 | 531 | 532 | 584 | 598 | 642 | |||
AAV1 | L | E | E | D | F | A | N | S | 70 |
AAV1-E/K | L | E | K | D | F | A | N | HS+ (and S)c | 70 |
AAV6 | F | D | K | D | L | V | H | HS and S | 70 |
AAV6.1 | F | D | E | D | L | V | H | HS− (and S)c | 40, 70 |
AAV6.2 | L | D | K | D | L | V | H | HS (and S)c | 40, 70 |
AAV6R2 | L | D | E | D | L | V | H | HS− (and S)c | 40 |
HAE1 | L | E | E | D | L | V | N | (HS− and S)d | 39 |
HAE2 | L | D | K | D | L | V | N | (HS and S)d | 39 |
shH10 | F | D | K | N | L | V | N | HS (and S-inde) | 33 |
20.
Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. 总被引:14,自引:0,他引:14
A Girod M Ried C Wobus H Lahm K Leike J Kleinschmidt G Deléage M Hallek 《Nature medicine》1999,5(9):1052-1056
The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy. 相似文献