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...

Adeno-associated virus (AAV) is a non-enveloped, icosahedral virus packaging a singlestranded DNA genome, and in recent years has garnered significant research interest for use as a human gene therapy vector due to its non-pathogenic nature. AAV expresses three overlapping structural proteins: VP1, VP2, and VP3, which assemble into a T=1 capsid (protein shell) at an approximate ratio of 1:1:10 ratio, and can package and deliver therapeutic transgenes to numerous cell types. However, little is known about the biology and biochemistry of how these viruses traffic through the endosomal/lysosomal pathway after receptor attachment and how this can affect the efficiency of transgene expression. Acidification in this pathway is essential for AAV infection, including the activation of a PLA2 enzyme encoded within their minor VP1 and localized inside of the capsid until required to function. Previous work from our lab has shown that AAV capsid VPs experience autolytic processing but complementary structural information is incomplete. Towards filling this gap, the structure of AAV serotype 2 (AAV2) has been determined by cryo-electron microscopy and 3D image reconstructions at the pH conditions experienced in the extracellular space, early endosome, late endosome, and lysosome, pH 7.4, 6.0, 5.5, and 4.0, respectively, to ~3.8 Å resolution. At this resolution, the amino acid side-chains were interpretable for the C-terminal ~520 residues of the VP3 common region, similar to previous reports for other AAVs and parvovirus capsids. The structures show surface loop re-arrangements concomitant with drop in pH while the core VP structure remains unchanged. We also show that capsid thermal stability increases with decreasing pH conditions. The functional implications of these observations will be discussed, especially within the context of the AAV life cycle and gene therapy vector design.