Molecular characterization of adeno-associated viruses infecting children

Abstract

Although adeno-associated virus (AAV) infection is common in humans, the biology of natural infection is poorly understood. Since it is likely that many primary AAV infections occur during childhood, we set out to characterize the frequency and complexity of circulating AAV isolates in fresh and archived frozen human pediatric tissues. Total cellular DNA was isolated from 175 tissue samples including freshly collected tonsils (n = 101) and archived frozen samples representing spleen (n = 21), lung (n = 16), muscle (n = 15), liver (n = 19), and heart (n = 3). Samples were screened for the presence of AAV and adenovirus sequences by PCR using degenerate primers. AAV DNA was detected in 7 of 101 (7%) tonsil samples and two of 74 other tissues (one spleen and one lung). Adenovirus sequences were identified in 19 of 101 tonsils (19%), but not in any other tissues. Complete capsid gene sequences were recovered from all nine AAV-positive tissues. Sequence analyses showed that eight of the capsid sequences were AAV2-like (approximately 98% amino acid identity), while the single spleen isolate was intermediate between serotypes 2 and 3. Comparison to the available AAV2 crystal structure revealed that the majority of the amino acid substitutions mapped to surface-exposed hypervariable domains. To further characterize the AAV capsid structure in these samples, we used a novel linear rolling-circle amplification method to amplify episomal AAV DNA and isolate infectious molecular clones from several human tissues. Serotype 2-like viruses were generated from these DNA clones and interestingly, failed to bind to a heparin sulfate column. Inspection of the capsid sequence from these two clones (and the other six AAV2-like isolates) revealed that they lacked arginine residues at positions 585 and 588 of the capsid protein, which are thought to be essential for interaction with the heparin sulfate proteoglycan coreceptor. These data provide a framework with which to explore wild-type AAV persistence in vivo and provide additional tools to further define the biodistribution and form of AAV in human tissues.

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.

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.

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.

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.

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.

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. C_α 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.

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.