Tyrosine Cross-Linking Reveals Interfacial Dynamics in Adeno-Associated Viral Capsids during Infection

Viral capsid dynamics are often observed during infectious events such as cell surface attachment, entry and genome release. Structural analysis of adeno-associated virus (AAV), a helper-dependent parvovirus, revealed a cluster of surface-exposed tyrosine residues at the icosahedral two-fold symmetry axis. We exploited the latter observation to carry out selective oxidation of Tyr residues, which yielded cross-linked viral protein (VP) subunit dimers, effectively “stitching” together the AAV capsid two-fold interface. Characterization of different Tyr-to-Phe mutants confirmed that the formation of cross-linked VP dimers is mediated by dityrosine adducts and requires the Tyr704 residue, which crosses over from one neighboring VP subunit to the other. When compared to unmodified capsids, Tyr-cross-linked AAV displayed decreased transduction efficiency in cell culture. Surprisingly, further biochemical and quantitative microscopy studies revealed that restraining the two-fold interface hinders externalization of buried VP N-termini, which contain a phospholipase A2 domain and nuclear localization sequences critical for infection. These adverse effects caused by tyrosine oxidation support the notion that interfacial dynamics at the AAV capsid two-fold symmetry axis play a role in externalization of VP N-termini during infection

Structural analysis of cysteine residues in the AAV2 capsid VP3 subunit.

<p>(a) Locations of cysteine residues on the major capsid subunit VP3 are shown in a capsid subunit pentamer. The VP3 subunits are colored pale yellow, light blue, pale green, wheat, and light pink and the side chains of each cysteine residue are highlighted using red spheres. (b) Close up view of the positions of unpaired Cys289 and Cys361 residues within a single subunit and (d) unpaired Cys230 and Cys394 residues at the interface between two VP3 subunits at the five-fold axis of symmetry. Thiol side chains are colored red and the two VP3 subunits in pale green and light blue. Low energy snapshots from DMD simulations demonstrating the feasibility of disulfide formation between Cys289/361 pair (c) and the Cys230/394 pair (e). All images were generated using Pymol®.</p

Probing the Link among Genomic Cargo, Contact Mechanics, and Nanoindentation in Recombinant Adeno-Associated Virus 2

Recombinant adeno-associated virus (AAV) is a promising gene therapy vector. To make progress in this direction, the relationship between the characteristics of the genomic cargo and the capsid stability must be understood in detail. The goal of this study is to determine the role of the packaged vector genome in the response of AAV particles to mechanical compression and adhesion to a substrate. Specifically, we used atomic force microscopy to compare the mechanical properties of empty AAV serotype 2 (AAV2) capsids and AAV2 vectors packaging single-stranded DNA or self-complementary DNA. We found that all species underwent partial deformation upon adsorption from buffer on an atomically flat graphite surface. Upon adsorption, a preferred orientation toward the twofold symmetry axis on the capsid, relative to the substrate, was observed. The magnitude of the bias depended on the cargo type, indicating that the interfacial properties may be influenced by cargo. All particles showed a significant relative strain before rupture. Different from interfacial interactions, which were clearly cargo-dependent, the elastic response to directional stress was largely dominated by the capsid properties. Nevertheless, small differences between particles laden with different cargo were measurable; scAAV vectors were the most resilient to external compression. We also show how elastic constant and rupture force data sets can be analyzed according a multivariate conditional probability approach to determine the genome content on the basis of a database of mechanical properties acquired from nanoindentation assays. Implications for understanding how recombinant AAV capsid–genome interactions can affect vector stability and effectiveness of gene therapy applications are discussed

Characterization of Cys-to-Ser mutants.

<p>Western dot blot of different fractions of wtAAV2 and Cys-to-Ser mutants resolved on a cesium chloride gradient. Aliquots 7* and 11* represent peak fractions with genome-containing particles and empty virion shells, respectively. Note the absence of A20 antibody staining for C230S and C394S mutants. Vector genome titers determined by Q-PCR are indicated adjacent to the dot blot.</p

Further mutagenesis of Cys230 and Cys394 residues.

<p>Transduction efficiency (luciferase expression) of transfected cell lysates containing a panel of (a) C230 mutants and (b) C394 mutants was evaluated at 24 hr post-transduction in HEK293 cells. Only the C394V mutant displays transduction efficiency similar to wtAAV2 vectors, while the corresponding C230V mutant is not viable. Error bars represent standard deviation (n = 3).</p

Proteasomal inhibition studies with wtAAV2, C230S and C394S mutant vectors.

<p>(a) Western blot analysis of capsid protein subunits in cell lysates using the B1 antibody at 18 hrs post-transfection with (+) or without (−) proteasome inhibitor, MG132. (b) Comparison of transduction efficiency (luciferase expression) of corresponding cell lysates shows that treatment with MG132 partially restores capsid protein expression, but not infectivity (light grey bars) when compared to control (dark grey bars). Error bars represent standard deviation (n = 3).</p

Optical Control of CRISPR/Cas9 Gene Editing

The CRISPR/Cas9 system has emerged as an important tool in biomedical research for a wide range of applications, with significant potential for genome engineering and gene therapy. In order to achieve conditional control of the CRISPR/Cas9 system, a genetically encoded light-activated Cas9 was engineered through the site-specific installation of a caged lysine amino acid. Several potential lysine residues were identified as viable caging sites that can be modified to optically control Cas9 function, as demonstrated through optical activation and deactivation of both exogenous and endogenous gene function

Schematic representation of the HBoV3 episome, results of inverse PCR and sequence alignment of HBoV genomic termini.

<p>(A) Describes the genomic organization of the HBoV3 episome and location/orientation of PCR primers used for screening samples and inverse PCR. (B) Shows results of inverse PCR assay for three samples (B-1 to B-3) that were positive for HBoV3 screening PCR with molecular weight (MW) marker and negative (Neg) reagent PCR control. (C) Shows alignment of previously known genomic termini of HBoV3 and HBoV1 linear genomes with the non-coding region of HBoV3 episome. Unique sequence identified in this study is shown as connecting the 3′ and 5′ termini (nucleotide with no background and “-” represent sequence that remained elusive in previous studies).</p

Comparative phylogenetic analysis of HBoV3-E1 and HBoV2-IB1 (filled rectangles).

<p>Nucleotide alignments were generated using the complete coding sequence of the NS, NP and VP genes. Names of sequences used for analysis are shown as Genbank accession numbers followed by the names of HBoV species and strains. The tree was constructed with the maximum likelihood method using a GTR+G substitution model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021362#pone.0021362-Tamura1" target="_blank">[38]</a>. Bootstrap replicates (>70%) are shown above the branches and distances (>0.02) are shown below the braches.</p

Secondary structure prediction of the sequences immediately downstream of the structural gene of different HBoV species (upper-HBoV1, middle-HBoV2 and lower-HBoV3-E1).

<p>The stems and arms are numbered for comparison, and the termination codon of the VP gene is marked by a solid line. The rabbit-ear structure (structure 3 and 4) present in all 3 HBoV species is comparable to similar conserved structures of left-hand side termini reported for animal bocaviruses (MVC and BPV) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021362#pone.0021362-Sun1" target="_blank">[44]</a>.</p