Halophilic viruses with varying biochemical and biophysical properties are amenable to purification with asymmetrical flow field-flow fractionation

Abstract

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 µm, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.

Controlled Disassembly and Purification of Functional Viral Subassemblies Using Asymmetrical Flow Field-Flow Fractionation (AF4)

“…The obtained yield and purity (specific infectivity value for the virus peak was ~9 × 10 11 PFU/A 280 ) were comparable to those previously reported [ 5 ]. Good recovery of prokaryotic viruses with various biophysical properties after AF4 purification has also been reported previously [ 38 , 39 ].…”

“…Our recent AF4-multi-angle light-scattering measurement (MALS) study showed that ultracentrifugation-based purification of φ6 yields virus specimen that has a relatively homogenous size distribution and little aggregates [ 5 ]. Virus-sized particles are generally well retained in the AF4 channel and elute at low cross-flow rates [ 5 , 38 , 39 , 45 ]. When compared to our previous study on φ6 that was performed with a thicker 350 μm spacer and resulted in virus elution at the end of the cross-flow gradient [ 5 ], here the thinner spacer promoted virus elution at higher cross-flow rates (peak maxima at a cross-flow rate of ~0.25 mL/min), providing improved resolution between monomeric viruses and putative larger sample components that are present if purification is performed with less purified inputs such as lysates or virus precipitates [ 5 ].…”

“…Analysis of the protein content of AF4 fractions by SDS-PAGE verified that P3 eluted at the beginning of the cross-flow gradient in the first fractions and was thus successfully released by the BHT-treatment ( Figure 2 B). The used low-flow rates were optimized for the separation of virus-sized macromolecular complexes, whereas soluble proteins that are smaller than ~700 kDa were expected to elute as a single peak at the beginning of the cross-flow gradient [ 38 , 39 ]. Based on the A 280 measurements, 62 ± 12% ( n = 7) of the BHT-treated virus was recovered in the major peak that eluted at the end of the cross-flow gradient.…”

“…The AF4 experiments and data collection were performed using an AF2000 MT instrument and Postnova AF2000 software (Postnova Analytics, Landsberg, Germany) as previously described [ 5 , 38 , 39 ], except that a teflon spacer of 250 μm and a regenerated cellulose (RC) membrane with a molecular weight cut-off (MWCO) value of 10 kDa (Postnova) were used (Lots 1551623 and 1653893). Channel flow was monitored in volts (V) at 260 nm (Shimadzu SPD-20A; Shimadzu, Kyoto, Japan).…”

Fast and Purification-Free Characterization of Bio-Nanoparticles in Biological Media by Electrical Asymmetrical Flow Field-Flow Fractionation Hyphenated with Multi-Angle Light Scattering and Nanoparticle Tracking Analysis Detection

“…Perpendicular to the laminar channel flow, a second flow (cross flow) and an electric field are applied simultaneously thereby inducing separation of sample constituents by size and charge ( Figure S1 ). Besides offering high-resolution separation and access to particle size distribution and surface charge (Zeta potential) of a respective sample, the possibility to purify (i.e., to remove matrix components smaller than the molecular weight cutoff of the ultrafiltration membrane via the cross flow) and separate matrix components from the analyte of interest renders EAF4 (just like AF4) a promising tool for an online purification of complex biological samples prior to further multi-detector analysis [ 19 , 20 ].…”