Study of rabies virus by Differential Scanning Calorimetry

Abstract

Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.

Thermal analysis of protein stability and ligand binding in complex media

“…Several published studies reported DSC analysis of a number of different viruses showing diagnostically unique thermograms that provide an additional means of their characterization, [28][29][30][31]. Unique thermograms for different viruses could be due to differences in the array of proteins that typically decorate viral coat surfaces [30].…”

Biophysical Characterization of Viral and Lipid-Based Vectors for Vaccines and Therapeutics with Light Scattering and Calorimetric Techniques

“…DSC data inform on unfolding of proteins and protein domains [44], thermotropic phase transitions of lipids [45], thermal transitions of RNA and DNA molecules [46][47][48] and thermally induced disassembly of viruses-the complex associations of nucleic acids with proteins and, in some cases, lipids [49]. DSC is used in development and manufacturing of several commercial vaccines [50][51][52][53][54] and informs research and development of lipid-based delivery vectors, their equivalence and similarity [19,54,55]. In addition to multiple stability metrics, DSC provides a fingerprint of Higher Order Structure defined by a range of intra-and inter-molecular interactions in a sample.…”

Bacterial Zombies and Ghosts: Production of Inactivated Gram-Positive and Gram-Negative Species with Preserved Cellular Morphology and Cytoplasmic Content

“…It acts mainly by damaging the DNA [6]. Previous research has found that BPL chemically modifies membrane fusion proteins and antigen proteins on the surface of Influenza viruses whereas another study showed that protein structures and foldings are not greatly affected in rabies viruses [7; 8; 9]. BPL has been previously applied on bacterial cells, but it is not known if BPL affects the bacterial surface structure.…”