Capillary Electrophoresis, Gas-Phase Electrophoretic Mobility Molecular Analysis, and Electron Microscopy: Effective Tools for Quality Assessment and Basic Rhinovirus Research

Abstract

We describe standard methods for propagation, purification, quality control, and physicochemical characterization of human rhinoviruses, using HRV-A2 as an example. Virus is propagated in HeLa-OHIO cells grown in suspension culture and purified via sucrose density gradient centrifugation. Purity and homogeneity of the preparations are assessed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), gas-phase electrophoretic mobility molecular analysis (GEMMA), and electron microscopy (EM). We also briefly describe usage of these methods for the characterization of subviral particles as well as for the analysis of their complexes with antibodies and soluble recombinant receptor mimics.

Analysis of a Common Cold Virus and Its Subviral Particles by Gas-Phase Electrophoretic Mobility Molecular Analysis and Native Mass Spectrometry

“…To remove the contaminant, we modified the conventional virus preparation protocol; 41 an additional lipase digestion step was included prior to the sucrose density gradient ultracentrifugation (see Materials and Methods section). As demonstrated in the GEMMA measurements depicted in Figure 2 a, the modified protocol yielded virus of exceptional purity; no contaminating material was detectable.…”

“…HRV-A2 was prepared either according to standard protocols 41 (two preparations) or, in the case of highly pure virus preparations, according to a slightly changed protocol including a lipase (porcine pancreas, Sigma-Aldrich) digestion step (two preparations). In short, HRV-A2 was grown in a HeLa-H1 cell suspension culture.…”

“…CE and TEM measurements were carried out as described elsewhere in detail. 41 An additional overview is given in the supplement.…”

“…Desalting and exchange of HRV-A2 stock storage buffer for NH 4 OAc was carried out as described by using 10 kDa M r cutoff spin filters (poly(ether sulfone) membrane from VWR, Vienna, Austria). 27 The HRV-A2 concentration of the desalted stock was calculated on the basis of (i) the initial virus concentration determined by CE, 41 (ii) sample dilution during desalting, and (iii) neglecting virus interaction with the membrane (which reduced the amount of a similar analyte by only roughly 20%). Hence, for simplification, a total recovery of the analyte from the filter was assumed.…”

Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site

“…Uncoating inhibition was confirmed by stabilization of RV-B5 by OBR-5-340 against temperature-dependent conversion into the permeable subviral A-particle, an uncoating intermediate. Since RV-B5 was found to grow poorly in spinner cultures, as employed for mass production of numerous other serotypes (38), we solved the 3D structure to 3.6 Å with the available small amounts of RV-B5 complexed to OBR-5-340 by using cryogenic electron microscopy (cryo-EM). For comparison, 3D structures of RV-B5 (in the absence of OBR-5-340) and of naturally drug-resistant RV-A89 (in the presence of OBR-5-340) were determined at 3.2 and 2.9 Å, respectively.…”