5ipk | pdb_00005ipk
Structure of the R432A variant of Adeno-associated virus type 2 VLP
Structural highlights
Function[CAPSD_AAV2S] Capsid protein self-assembles to form an icosahedral capsid with a T=1 symmetry, about 22 nm in diameter, and consisting of 60 copies of three size variants of the capsid protein VP1, VP2 and VP3 which differ in their N-terminus. The capsid encapsulates the genomic ssDNA. Binds to host cell heparan sulfate and uses host ITGA5-ITGB1 as coreceptor on the cell surface to provide virion attachment to target cell. This attachment induces virion internalization predominantly through clathrin-dependent endocytosis. Binding to the host receptor also induces capsid rearrangements leading to surface exposure of VP1 N-terminus, specifically its phospholipase A2-like region and putative nuclear localization signal(s). VP1 N-terminus might serve as a lipolytic enzyme to breach the endosomal membrane during entry into host cell and might contribute to virus transport to the nucleus.[1] [2] [3] [4] Publication Abstract from PubMedThe 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 approximately 5.0 A (medium) and also 3.8 and 3.7 A (high) resolution, 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 towards the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an approximately 10 degrees C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. Additionally, 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 betaA strand region under the icosahedral 2-fold axis rather than anti-parallel to the betaB 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. IMPORTANCE: The 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 approximately 1:10 (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. Cryo-electron microscopy reconstruction and stability studies of Wild-Type and R432A Variant of AAV2 Reveals Capsid Structural Stability is a Major Factor in Genome Packaging.,Drouin LM, Lins B, Janssen M, Bennett A, Chipman P, McKenna R, Chen W, Muzyczka N, Cardone G, Baker TS, Agbandje-McKenna M J Virol. 2016 Jul 20. pii: JVI.00575-16. PMID:27440903[5] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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