Asymmetrical flow field-flow fractionation in virus purification

Viruses are the most abundant entities in the biosphere, the estimated amount of viruses is more than 10^30. The number is incomprehensible and exceeds the amount of host cells at least by one order of magnitude. Viruses are extremely diverse entities by means of morphologies, sizes, genomes and biochemical and biophysical properties. As obligate parasites, viruses can only be propagated in living cells. This sets challenges for the virus purification, since the starting material contains host and growth media derived impurities. Medical applications such as phage therapy, vaccine development, and gene therapy require large amounts of highly purified viruses and virus-like particles (VLPs). Nanotechnology utilizes viruses and VLPs as building blocks for nanoscale materials and devices and also requires virus purification methods which maintain the biophysical and biochemical properties of the particles. Viruses are often purified with combinations of different methods. The most common ones are precipitation and ultracentrifugation. Precipitation does not lead into high purities and is generally applied as a pre-step for purification. Ultracentrifugation leads to high purity but it exposes viruses to high shear forces possibly leading to losses of infectivity. The large size of many viruses may restrict utilization of traditional chromatography. However, monolithic matrices are applicable for virus purification. In this work asymmetrical flow field-flow fractionationation (AF4) method was developed for virus purification. AF4 is a highly versatile size-based separation method applicable for samples with sizes ranging between ~1‒500 nm. The separation in AF4 is conducted with the aid of liquid flows. Solid stationary phase is not applied at all, thus no strong interactions during the separation occur making the method gentle. Several parameters in the AF4 system are adjustable, making the method highly versatile and an attractive alternative for virus purification. In this study, AF4 conditions were optimized for purification of six prokaryotic viruses, having different morphologies and properties. Analytical sample channel and preparative UV-detector were utilized. Yields of infective viruses were high and purity levels comparable to the ones obtained with a method based on precipitation and ultracentrifugation. AF4 was proven to be applicable for all tested viruses, also the ones requiring high ionic strength conditions were amenable for AF4 purification. The AF4-method is fast and obtained virus preparations were homogenous. As the system is highly versatile, it is expected that it can be tailored for other viruses as well, to meet the further needs of virus purification.Virusten määrä ympäristössämme on tähtitieteellinen. Viruspartikkeleita on arvioitu olevan biosfäärissä jopa 10^30 kappaletta, näinollen ylittäen isäntäsolujen määrän jopa kymmenkertaisesti. Virukset ovat hyvin kirjava joukko niin morfologioiden, kokojen, genomien kuin biokemiallisten ja – fysikaalisten ominaisuuksiensa suhteen. Kuitenkin, virukset voivat lisääntyä ainoastaan elävissä soluissa. Tämä asettaa omat haasteensa viruspartikkelien puhdistamiselle, sillä lähtömateriaali sisältää isäntäsolusta ja kasvatusalusta peräisin olevia epäpuhtauksia. Lääketieteen sovellutukset, kuten faagiterapia, rokotekehitys ja geeniterapia tarvitsevat suuria määriä korkean puhtaustason viruksia ja viruksen kaltaisia partikkeleita. Nanoteknologiassa korkean puhtaustason viruksia ja viruksen kaltaisia partikkeleita käytetään rakennuspalikoina nanomittaluokan materiaaleissa. Lääketiede, nanoteknologia ja muut viruksia hyödyntävät alat tarvitsevat nopeita ja tehokkaita viruspuhdistusmenetelmiä, jotka säilyttävät viruksen ominaisuudet ja johtavat korkeisiin saantoihin. Useimmiten viruspuhdistuksessa yhdistellään eri menetelmiä. Yleisimpiä näistä menetelmistä ovat saostus ja ultrasentrifugaatio. Saostus ei yksin johda korkeaan puhtaustasoon ja sitä käytetäänkin tavallisesti puhdistuksen ensimmäisenä askeleena. Ultrasentrifugoinnilla sen sijaan saavutetaan korkea puhtausaste, mutta altistetaan virukset koville sentrifugointivoimille. Tämä saattaa johtaa partikkelien vahingoittumiseen ja infektiivisyyden menettämiseen. Virukset ovat makromolekulaarisia komplekseja, joten niiden koko rajoittaa useiden perinteisten kromatografiamenetelmien käyttöä niiden puhdistuksessa. Tässä työssä kehitettiin viruspuhdistusmenetelmä asymmetrista virtauskenttäfraktiointia (AF4) hyödyntäen. AF4 on hyvin monipuolinen, kokoon perustuva erottelumenetelmä näytteille kokoluokassa ~1‒500 nm. Erottelu saadaan aikaiseksi nestevirtauksilla eikä menetelmässä käytetä lainkaan kiinteä stationäärifaasia. AF4-erottelu onkin erityisen hellävarainen menetelmä, sillä voimakkaita vuorovaikutuksia näytteen ja laitteiston välillä ei synny. Lukuisat AF4-laitteiston ominaisuudet ovat säädettävissä näytteen erityispiirteille sopiviksi, tehden menetelmästä houkuttelevan vaihtoehdon viruspuhdistuksessa. Tässä projektissa AF4-olosuhteet optimoitiin viruspuhdistuksen tarpeisiin kuutta prokaryoottivirusta hyödyntäen. Optimointiin käytetyt virukset edustivat eri morfologiatyyppejä hyvin erilaisine ominaisuuksineen. Laitteistossa käytettiin analyyttista näytekanavaa ja preparatiivista UV-detektoria. Virussaannot olivat korkeita ja puhtaustaso vastasi saostuksen ja ultrasentrifugoinnin yhdistelmällä saavutettavaa puhtautta. Osoitimme AF4-menetelmän soveltuvan kaikille testatuille viruksille, myös niille, jotka vaativat korkeita suolapitoisuuksia säilyttääkseen infektiivisyytensä. AF4-menetelmä on nopea ja puhdistetut viruspreparaatit laadultaan homogeenisia. Perinteinen, saostukseen ja ultrasentrifugointiin pohjautuva puhdistusmenetelmä on työläs ja aikaa vievä. AF4-menetelmällä puhdistusaika lyheni merkittävästi ja korkeiden saantojen vuoksi lähtömateriaalin tarve väheni moninkertaisesti. Koska AF4-menetelmä on helposti muokattavissa, sen voi olettaa soveltuvan myös muille kuin työssä testatuille viruksille ja eri tieteenalojen viruspuhdistuksen tarpeisiin

Native RNA purification method for small RNA molecules based on asymmetrical flow field-flow fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules.Peer reviewe

Analysis and purification of ssRNA and dsRNA molecules using asymmetrical flow field flow fractionation

Robust RNA purification and analysis methods are required to support the development of RNA vaccines and therapeutics as well as RNA interference-based crop protection solutions. Asymmetrical flow field -flow fractionation (AF4) is a gentle native purification method that applies liquid flows to separate sample components based on their hydrodynamic sizes. We recently showed that AF4 can be utilized to separate RNA molecules that are shorter than 110 nucleotides (nt), but the performance of AF4 in the analysis and purification of longer RNA molecules has not been previously evaluated. Here, we studied the perfor-mance of AF4 in separation of single-stranded (ss) and double-stranded (ds) RNA molecules in the size range of 75-6400 nt. In addition, we evaluated the power of AF4 coupling to different detectors, allow-ing separation to be combined with data collection on yield as well as molecular weight ( MW ) and size distribution. We show that AF4 method is applicable in RNA purification, quality control, and analytics, and results in good recoveries of ssRNA and dsRNA molecules. In addition, our results demonstrate the utility of AF4 multidetection platforms to study biophysical properties of long RNA molecules.(c) 2022 The Author(s). Published by Elsevier B.V.This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ )Peer reviewe

Bacteriophage Infection of the Marine Bacterium Shewanella glacialimarina Induces Dynamic Changes in tRNA Modifications

Viruses are obligate intracellular parasites that, throughout evolution, have adapted numerous strategies to control the translation machinery, including the modulation of post-transcriptional modifications (PTMs) on transfer RNA (tRNA). PTMs are critical translation regulators used to further host immune responses as well as the expression of viral proteins. Yet, we lack critical insight into the temporal dynamics of infection-induced changes to the tRNA modification landscape (i.e., ‘modificome’). In this study, we provide the first comprehensive quantitative characterization of the tRNA modificome in the marine bacterium Shewanella glacialimarina during Shewanella phage 1/4 infection. Specifically, we show that PTMs can be grouped into distinct categories based on modification level changes at various infection stages. Furthermore, we observe a preference for the UAC codon in viral transcripts expressed at the late stage of infection, which coincides with an increase in queuosine modification. Queuosine appears exclusively on tRNAs with GUN anticodons, suggesting a correlation between phage codon usage and PTM modification. Importantly, this work provides the basis for further studies into RNA-based regulatory mechanisms employed by bacteriophages to control the prokaryotic translation machinery

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

Asymmetric flow field flow fractionation methods for virus purification

Detailed biochemical and biophysical characterization of viruses requires viral preparations of high quantity and purity. The optimization of virus production and purification is an essential, but laborious and time-consuming process. Asymmetric flow field flow fractionation (AF4) is an attractive alternative method for virus purification because it is a rapid and gentle separation method that should preserve viral infectivity. Here we optimized the AF4 conditions to be used for purification of a model virus, bacteriophage PRD1, from various types of starting materials. Our results show that AF4 is well suited for PRD1 purification as monitored by virus recovery and specific infectivity. Short analysis time and high sample loads enabled us to use AF4 for preparative scale purification of PRD1. Furthermore, we show that AF4 enables the rapid real-time analysis of progeny virus production in infected cells. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Asymmetrical flow field-flow fractionation in purification of an enveloped bacteriophage φ 6

Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage phi 6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of phi 6. In addition, binding of phi 6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of similar to 1 x 10(13) PFU/mg of protein and similar to 65-95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2-3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with inline light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.Peer reviewe

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

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

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 A mu m, 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.Peer reviewe

Bacteriophage Infection of the Marine Bacterium Shewanella glacialimarina Induces Dynamic Changes in tRNA Modifications

Viruses are obligate intracellular parasites that, throughout evolution, have adapted numerous strategies to control the translation machinery, including the modulation of post-transcriptional modifications (PTMs) on transfer RNA (tRNA). PTMs are critical translation regulators used to further host immune responses as well as the expression of viral proteins. Yet, we lack critical insight into the temporal dynamics of infection-induced changes to the tRNA modification landscape (i.e., ‘modificome’). In this study, we provide the first comprehensive quantitative characterization of the tRNA modificome in the marine bacterium Shewanella glacialimarina during Shewanella phage 1/4 infection. Specifically, we show that PTMs can be grouped into distinct categories based on modification level changes at various infection stages. Furthermore, we observe a preference for the UAC codon in viral transcripts expressed at the late stage of infection, which coincides with an increase in queuosine modification. Queuosine appears exclusively on tRNAs with GUN anticodons, suggesting a correlation between phage codon usage and PTM modification. Importantly, this work provides the basis for further studies into RNA-based regulatory mechanisms employed by bacteriophages to control the prokaryotic translation machinery.Peer reviewe