Comparative Analysis of Adeno-Associated Virus Capsid Stability and Dynamics

Abstract

bIcosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.

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

“…8). This observation is congruent with a previously reported T m of 67.8 Ϯ 0.2°C (59). In contrast, the AAV2-R432A melting profile exhibited a "shoulder" prior to the major peak, resulting in two melting temperatures: peak 1 ϭ 57.5 Ϯ 0.2°C and peak 2 ϭ 61.1 Ϯ 1.9°C (Fig.…”

“…In addition, the mutant AAV displayed two melting peaks, suggesting dual denaturation events. Of note, the melting profile of AAV8 also demonstrates two transitions, separated by ϳ4.3°C (59). A plausible explanation for the AAV2-R432A melting behavior is that two populations of capsids exist, for example, one with altered ␤A conformation (as observed) and another in transition from the wt-AAV2 conformation.…”

“…Prior DSC studies have demonstrated that the specific heat capacity of the AAV capsid directly correlates to its structural stability, and so it follows that a lower T m indicates a capsid with decreased stability (59). In addition, the mutant AAV displayed two melting peaks, suggesting dual denaturation events.…”

“…Density consistent with the conformational change was seen only at a low sigma threshold (0.5 ) in the high-resolution map and when resolution was reduced to 5 Å, suggesting a reduced occupancy for the N-terminal amino acids observed beyond position 236. It is also possible that the reduced stability of AAV2-R432A results in dissociation into an intermediate VP oligomeric state such as trimers or pentamers prior to complete capsid disassembly, which appears to be a one step process for AAV2 and other serotypes (59).…”

Parvovirus Capsid Structures Required for Infection: Mutations Controlling Receptor Recognition and Protease Cleavages

“…Small differences in the structures and dynamics of various parvovirus and adeno-associated virus (AAV) capsids have been reported (35,47,48). Although the crystal structures of empty particles or virus-like particles (VLPs) appear structurally highly similar to those of infectious DNA-containing capsids when different crystal structures are compared, some differences in the structures and dynamics have been revealed (49)(50)(51).…”