Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

Abstract

The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ϳ5.0-Å resolution (medium) and also at 3.8-and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ϳ10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the ␤A strand region under the icosahedral 2-fold axis rather than antiparallel to the ␤B strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. IMPORTANCEThe mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ϳ1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.T he adeno-associated viruses (AAVs) have emerged as attractive gene therapy vectors because they can package foreign genes and achieve stable, long-term gene expression in a broad range of tissues, with no known pathogenicity (1-3). AAVs are small, nonenveloped, icosahedral viruses (ϳ260 Å in diameter) that package ϳ4.7 kb of single-stran...

An ancient lineage of highly divergent parvoviruses infects both vertebrate and invertebrate hosts

“…Thus, these positions could constitute potential determinants of tissue tropism in murine ChPVs. Parvoviruses subfamilies Parvovirinae and Densovirinae utilize distinct strategies to stabilize their icosahedral capsids (35). Vertebrate parvoviruses extend the longer side of the jellyroll fold with an additional, N-terminal strand by folding back β-A to interact with the twofold axis of the very same monomer, hence creating an extended ABDIG sheet (36, 37).…”

Sub-2 Å Ewald curvature corrected structure of an AAV2 capsid variant

“…The harvested pellet (from lysed cells and polyethylene glycol precipitated supernatant) was freeze/thawed three times with Benzonase (EMD Millipore Cat#712053) treatment. After the third thaw, the resulting clarified supernatant was purified using a step iodixanol gradient followed by anion exchange 46 and then dialyzed into 50 mM HEPES, pH 7.4 with 2 mM MgCl 2 and 150 mM NaCl. The sample concentration was determined by optical density assuming an extinction coefficient of 1.7 mg mL −1 cm −1 ) for AAV2 VLPs.…”

Evolution of a designed protein assembly encapsulating its own RNA genome

“…Trimeric subunits are colored green and pentameric subunits are colored cyan. Mutations with respect to the original I53-50 protein assembly 7 are colored blue (increases positive charge and/or decreases negative charge [e.g., E→N, N→K, E→K]), orange (no change in charge [e.g., E→D, N→T, K→R]), or red (decreases positive charge and/or increases negative charge [e.g., N→E, K→N, K→E]). b .…”

“…Several generations of evolution resulted in drastically improved genome packaging (>133-fold), stability in whole murine blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus (AAV) vectors 7, 8 . Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection.…”