Abstract
For vaccine development, 3D-structure determination, direct fluorescent labelling, and numerous other studies, homogeneous virus preparations of high purity are essential. Working with human rhinoviruses (RVs), members of the picornavirus family and the main cause of generally mild respiratory infections, we noticed that our routine preparations appeared highly pure on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), exclusively showing the four viral capsid proteins (VPs). However, the preparations turned out to contain substantial amounts of contaminating material when analyzed by orthogonal analytical methods including capillary zone electrophoresis, nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA), and negative stain transmission electron microscopy (TEM). Because these latter analyses are not routine to many laboratories, the above contaminations might remain unnoticed and skew experimental results. By using human rhinovirus serotype A2 (RV-A2) as example we report monolithic anion-exchange chromatography (AEX) as a last polishing step in the purification and demonstrate that it yields infective, highly pure, virus (RV-A2 in the respective fractions was confirmed by peptide mass fingerprinting) devoid of foreign material as judged by the above criteria.
Highlights
Picornaviruses are icosahedral particles of 30 nm diameter with a single-stranded (+) RNA genome enclosed within a non-enveloped protein shell composed of 60 copies each of the four capsid proteins VP1 through VP4
Working with human rhinovirus (RV), which belong to the genus Enteroviruses, and cause more than 50% of all respiratory infections, we noticed that routine preparations showed exclusively the four viral capsid proteins (VPs) upon sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
We demonstrate that anion-exchange chromatography (AEX) yields a pure rhinovirus serotype A2 (RV-A2) fraction well separated from other non-viral components, including ferritin and proteasomes, and free of the ‘contaminant’ as shown by negative stain transmission electron microscopy (TEM) and nano electrospray (nES) gas-phase electrophoretic mobility molecular analyzer (GEMMA)
Summary
Picornaviruses are icosahedral particles of 30 nm diameter with a single-stranded (+) RNA genome enclosed within a non-enveloped protein shell composed of 60 copies each of the four capsid proteins VP1 through VP4. Kremser et al (Kremser et al, 2006) already demonstrated, by using capillary electrophoresis with UV absorbance detection (CE-UV) that RV-A2 purified via ultracentrifugation on a sucrose density gradient contained some additional material (‘contaminant’). This material showed up as peak monitored at 200 nm UV absorption (typical wavelength for RV-A2 analysis applied in CE separation) in CE runs in presence of SDS above its critical micellar concentration in the background electrolyte (Kremser et al, 2006). When labelling RVA2 with amine-reactive NHS-ester dyes, the contaminant became strongly fluorescent as seen in CE with laser induced fluorescence detection (CE-LIF), suggesting either the presence of many primary amino groups or non-covalent attachment of the dye to hydrophobic surface patches or pockets of the contaminant (Kolivoska et al, 2007; Kremser et al, 2004; Weiss et al, 2007)
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