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Genome Replication and Regulation of Viral Gene Expression

Molecular Characterization of Adeno-Associated Viruses Infecting Children

Chun-Liang Chen, Ryan L. Jensen, Bruce C. Schnepp, Mary J. Connell, Richard Shell, Thomas J. Sferra, Jeffrey S. Bartlett, K. Reed Clark, Philip R. Johnson
Chun-Liang Chen
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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Ryan L. Jensen
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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Bruce C. Schnepp
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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Mary J. Connell
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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Richard Shell
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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Thomas J. Sferra
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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Jeffrey S. Bartlett
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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K. Reed Clark
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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Philip R. Johnson
Center for Gene Therapy, Columbus Children's Research Institute, Columbus Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205
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  • For correspondence: johnsonphi@chop.edu
DOI: 10.1128/JVI.79.23.14781-14792.2005
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  • FIG. 1.
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    FIG. 1.

    PCR schematic for amplification of the complete AAV capsid coding region. (A) The diagram depicts the relative location of degenerate primers (given in Materials and Methods) used to amplify the AAV cap gene. Initially samples were screened with degenerate nested primers (Cap18S, Cap19S, CapSS3189, CapSS2978) to two conserved regions that flank the HVR3 coding region (gray box). To amplify the complete capsid gene, another set of nested primers were constructed (AAV2-1.8F1, AAV2-1.8F2, AAVCap3′Rev, AAVCap3′RevDeg) that bind to 3′ regions of rep and cap and amplify 1.8- and 1.5-kb DNA amplicons. (B) Representative amplification of the 255-bp conserved AAV sequence from human tissue DNA (100 ng) following nested PCR (see Materials and Methods for reaction conditions). Asterisks indicate the samples that are positive for AAV amplification.

  • FIG. 2.
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    FIG. 2.

    Predicted VP1 capsid amino acid alignment of AAV2 and novel human AAVs. Diagram shows sequence alignment using the CLUSTAL W program. Black boxes designate amino acid substitutions compared to the AAV2 sequence. The locations of previously identified HVR regions (11) are labeled (HVR 1 to 12), as is an additional region (HVR 2′) that possesses several substitutions. Several HRV regions (5 to 7, 9, and 10) are colored to facilitate visualization of these regions onto the known atomic structure of AAV2, while invariant HVRs are labeled with black boxes. The locations of R585S and R588T are starred, and arrows denote the approximate locations of nested primers used to amplify the 255-bp HVR3 fragment.

  • FIG. 3.
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    FIG. 3.

    Phylogenetic analysis of VP1 capsid nucleotide sequences. A neighbor-joining program with a Kimura two-parameter setting was used to derive phylogenetic distances based on 2,200 bp of VP1 sequence. Recently described AAV clade nomenclature (12) was adopted and organized by vertical brackets. The human isolates identified herein are designated in teal type. Due to space restrictions, only a few representative isolates from clades A, D, and E are shown. Sequence isolates are labeled with reference to the source species (bb, baboon; ch, chimpanzee; cy, cynomolgus macaque; hu, human; rh, rhesus macaque). Clade B sequences possessing R585 and R588 amino acids and predicted to bind HSPG efficiently are labeled in red type. The scale for genetic distance is indicated in the bottom left corner.

  • FIG. 4.
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    FIG. 4.

    S17 sequence homology comparison with AAV2 and AAV3. Simplot analyses of similarity percentages of S17 VP1 versus AAV 2 (red) and AAV3 (blue) are shown. Data were plotted within a sliding window of 200 bp, centered on the position plotted, with a step size between data points of 20 bp. Positions containing gaps were excluded from the comparison. The bar on the top shows the predicted composition of the S17 capsid gene. The corresponding positions of the HVRs are labeled as magenta boxes (HVR 2′ in gray).

  • FIG. 5.
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    FIG. 5.

    Surface diagrams of AAV2 trimer atomic models. (A) Electrostatic surface potential of the VP3 AAV2 trimer viewed down the threefold axis (yellow triangle) calculated with GRASP (23) running from negative (red) to positive (blue). Labeled arrows indicate the positions of residues implicated in HSPG binding. (B) Predicted electrostatic surface potential of AAV2 VP3 trimer as a result of R585S and R588T substitutions. Amino acid substitutions were modeled using energy minimization simulations with Quanta (Accelrys, San Diego, CA) prior to generating the electrostatic potential map in GRASP. The surface electrostatic potential scale is the same as depicted in panel A. Highlighted regions denote predicted HSPG coreceptor engagement domains in the VP3 trimer.

  • FIG. 6.
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    FIG. 6.

    Ribbon diagrams of atomic models of AAV2 VP3 trimers showing the location of predicted amino acid substitutions in the human AAV isolates. (A) Ribbon drawing viewed down the threefold axis of symmetry of the AAV2 VP3 trimer. Cα backbones for the three VP3 monomers are rendered as teal ribbons. Predicted locations of the observed amino acid substitutions present within the eight AAV2-like sequences are color coded to reflect HVR location (HVR 5 to 7, 9, and 10) within the primary sequence (Fig. 2). White space-filling amino acid substitutions mapped outside the known HVRs. (B) Side view of the predicted location of the observed amino acid substitution demonstrating surface display (right side). (C) Superimposition of observed S17 amino acid substitutions relative to the AAV2 VP3 trimer atomic structure viewed down the threefold axis. (D) Side view of the predicted location of the observed amino acid substitutions in isolate S17 (surface display oriented on right side). Images were generated in NAMD/VMD (UIUC Theoretical Biophysics Group) and rendered using Raster3D.

  • FIG. 7.
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    FIG. 7.

    Serology of AAV infection as a function of age. OD450 values from a standard ELISA (see Materials and Methods) are plotted versus the age of the subject. Sera were tested at a 1:100 dilution. OD values below 0.2 (thin solid line) were considered negative. The same sera were tested for neutralization activity against AAV2 (see Materials and Methods). Data points that are circled represent samples that had neutralization titers of >1:100.

Tables

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  • TABLE 1.

    Summary of AAV and adenovirus sequence detection in human tissuesa

    Viral DNA foundNo. of samples positive
    T+A (n = 101)Liver (n = 19)Spleen (n = 21)Muscle (n = 15)Heart (n = 3)Lung (n = 3)
    AAV positive701001
    AdV positive1900000
    AAV and AdV positive2b00000
    • ↵ a T+A = tonsils and adenoids; AdV = adenovirus.

    • ↵ b Two samples (T17 and T32) contained both AAV and adenovirus sequences.

  • TABLE 2.

    AAV sequence relatedness and DNA copy number in pediatric tissues

    Sample (age, yr)aAmino acid identityb (%)Nucleotide identityc (%)AAV copies/μgd
    T17 (2.3)AAV2 (98.1)AAV2 (96.0)560
    T32 (5.5)AAV2 (98.1)AAV2 (96.0)210
    T40 (2.9)AAV2 (98.0)AAV2 (96.1)6,550
    T41 (2.6)AAV2 (98.0)AAV2 (96.0)590
    T70 (3.2)AAV2 (98.4)AAV2 (96.8)7,800
    T71 (5.8)AAV2 (98.0)AAV2 (96.0)7,600
    T88 (4.7)AAV2 (98.0)AAV2 (96.5)33,000
    S17 (8)AAV3 (92.7)AAV2 (90.0)200
    LG15 (1)AAV2 (98.4)AAV2 (96.8)80
    • ↵ a T = tonsil/adenoid; S = spleen; LG = lung. The age of the subject in years is in parentheses.

    • ↵ b Percent amino acid identity to the indicated AAV serotype is given in parentheses.

    • ↵ c Percent nucleotide identity to the indicated AAV serotype is given in parentheses.

    • ↵ d AAV DNA copy number determined using Q-PCR (see Materials and Methods). Values shown are the average of two separate determinations.

  • TABLE 3.

    Heparin sulfate column binding of AAV2 virus preparations

    VirusNonbound DRPaBound DRPbPercent Bound
    AAV22.4 × 1091.5 × 10100.86
    T70-432.0 × 10101.0 × 1090.05
    T88-411.7 × 10101.4 × 1090.08
    • ↵ a DNase-resistant particle (DRP) copy number was determined using Taqman Q-PCR. Primers and probe were homologous to a conserved cap sequence (see Materials and Methods). Nonbound represent total virus found in wash and flowthrough material. Data shown are the average of two experiments.

    • ↵ b Copy number present in pooled 1-ml gradient fractions. Data shown are the average of two experiments.

  • TABLE 4.

    Summary of synonymous and nonsynonymous nucleotide substitutions in rep and cap

    IsolateSubstitutionsa
    cap HVR (400 bp)cap non-HVR (1,800 bp)3′ rep (600 bp)
    snss/nsRate/ntsnss/nsRate/ntsnss/nsRate/nt
    LG1520111.87.55769.53.5942.32.1
    S1748580.825.892910.25.61125.52.3
    T1717101.76.662415.53.7732.31.4
    T3217101.76.662415.53.7632.01.2
    T4012101.25.454318.03.2933.01.9
    T411581.95.647411.82.8431.31.1
    T701061.73.94868.03.01744.32.6
    T711581.95.647411.82.8331.00.9
    T881892.06.645592.81343.32.4
    Avg ± SD1.6 ± 0.278.2 ± 3.9b12.1 ± 2.8c3.5 ± 0.62.8 ± 1.11.8 ± 0.6
    • ↵ a s is the number of observed synonymous substitutions in the indicated coding region; ns is the number of observed nonsynonymous substitutions in the indicated coding region; s/ns is the calculated ratio. The rate/nt value represents the number of observed nucleotide substitutions per 100 bp of sequence.

    • ↵ b Substitution rate in HVR region was greater than that observed for non-HVR and 3′ rep region, P = 0.04 and 0.02, respectively, using paired t test.

    • ↵ c Ds/Da ratio in non-HVR region was greater than that observed for HVR and 3′ rep region, P = 0.00002 and 0.0001, respectively, using paired t test.

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Molecular Characterization of Adeno-Associated Viruses Infecting Children
Chun-Liang Chen, Ryan L. Jensen, Bruce C. Schnepp, Mary J. Connell, Richard Shell, Thomas J. Sferra, Jeffrey S. Bartlett, K. Reed Clark, Philip R. Johnson
Journal of Virology Nov 2005, 79 (23) 14781-14792; DOI: 10.1128/JVI.79.23.14781-14792.2005

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Molecular Characterization of Adeno-Associated Viruses Infecting Children
Chun-Liang Chen, Ryan L. Jensen, Bruce C. Schnepp, Mary J. Connell, Richard Shell, Thomas J. Sferra, Jeffrey S. Bartlett, K. Reed Clark, Philip R. Johnson
Journal of Virology Nov 2005, 79 (23) 14781-14792; DOI: 10.1128/JVI.79.23.14781-14792.2005
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KEYWORDS

Capsid Proteins
Dependovirus
Parvoviridae Infections

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