Fluorometric Estimation of Viral Thermal Stability.

Differential Scanning Fluorimetry (DSF) is a rapid, economical, and a straightforward technique for estimating the thermal stability of proteins. The principle involves the binding of a fluorescent dye to thermally exposed hydrophobic pockets of a protein. The dyes used in this technique are highly fluorescent in a non-polar environment and are quenched when exposed to aqueous solution. The change in fluorescence can be used to follow unfolding of proteins induced by temperature, pH, or chaotropic agents. The method is well characterized for monomeric proteins. Here, we extend the application to supramolecular protein and nucleo-protein complexes using virus particles as an example. SYPRO-orange™ dye is the dye of choice because it is matched for use with q-PCR instruments and the fluorescence response is stable across a wide range of pH and temperatures. Advantages of this technique over standard biophysical methods include the ability for high-throughput screening of biological and technical replicates and the high sensitivity.

Comparative analysis of adeno-associated virus capsid stability and dynamics.

Icosahedral 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.

Thermodynamic characterization of viral procapsid expansion into a functional capsid shell.

The assembly of "complex" DNA viruses such as the herpesviruses and many tailed bacteriophages includes a DNA packaging step where the viral genome is inserted into a preformed procapsid shell. Packaging triggers a remarkable capsid expansion transition that results in thinning of the shell and an increase in capsid volume to accept the full-length genome. This transition is considered irreversible; however, here we demonstrate that the phage λ procapsid can be expanded with urea in vitro and that the transition is fully reversible. This provides an unprecedented opportunity to evaluate the thermodynamic features of this fascinating and essential step in virus assembly. We show that urea-triggered expansion is highly cooperative and strongly temperature dependent. Thermodynamic analysis indicates that the free energy of expansion is influenced by magnesium concentration (3-13 kcal/mol in the presence of 0.2-10 mM Mg(2+)) and that significant hydrophobic surface area is exposed in the expanded shell. Conversely, Mg(2+) drives the expanded shell back to the procapsid conformation in a highly cooperative transition that is also temperature dependent and strongly influenced by urea. We demonstrate that the gpD decoration protein adds to the urea-expanded capsid, presumably at hydrophobic patches exposed at the 3-fold axes of the expanded capsid lattice. The decorated capsid is biologically active and sponsors packaging of the viral genome in vitro. The roles of divalent metal and hydrophobic interactions in controlling packaging-triggered expansion of the procapsid shell are discussed in relation to a general mechanism for DNA-triggered procapsid expansion in the complex double-stranded DNA viruses.