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High initial sputter rate found for Vaccinia virions using isotopic labeling, NanoSIMS and AFM Sean Damien Gates, Richard C Condit, Nissin Moussatche, Benjamin J. Stewart, Alexander J Malkin, and Peter K. Weber Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02786 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018
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Analytical Chemistry
High initial sputter rate found for Vaccinia virions using isotopic labeling, NanoSIMS and AFM Sean D. Gates1, Richard C. Condit2, Nissin Moussatche2, Benjamin J. Stewart3, Alexander J. Malkin3*, Peter K. Weber1* 1Nuclear
and Chemical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94551 2Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL 32610, 3Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, CA 94551 *Corresponding authors: Alexander J. Malkin, 7000 East Ave., L-233, Livermore, CA 94551, (tel.): 925-423-781;
[email protected]; Peter K. Weber, 7000 East Ave., L-231, Livermore, CA 94551, (tel): 925-422-3018;
[email protected] 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
ABSTRACT High lateral resolution secondary ion mass spectrometry (SIMS) has the potential to provide functional and depth resolved information from small biological structures, such as viral particles (virions) and phage, but sputter rate and sensitivity are not characterized at shallow depths relevant to these structures. Here we combine stable isotope labeling of the DNA of vaccinia virions with correlated SIMS imaging depth profiling and atomic force microscopy (AFM) to develop a non-linear, non-equilibrium sputter rate model for the virions and validate the model based on reconstructing the location of the DNA within individual virions. Our experiments with a Cs+ beam show an unexpectedly high initial sputter rate (~100 um2·nm·pA-1·s-1) with a rapid decline to an asymptotic rate of 0.7 um2·nm·pA-1·s-1 at an approximate depth of 70 nm. Correlated experiments were also conducted with glutaraldehyde-fixed virions, as well as O- and Ga+ beams, yielding similar results. Based on our Cs+ sputter rate model, the labeled DNA in the virion was between 50 and 90 nm depth in the virion core, consistent with expectations, supporting our conclusions. Virion densification was found to be a secondary effect. Accurate isotopic ratios were obtained from the initiation of sputtering, suggesting that isotopic tracers could be successfully used for smaller virions and phage.
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INTRODUCTION Due to their size, lack of symmetry (e.g., icosahedral, helical), structural heterogeneity, and high molecular weight, most large animal and human viruses, including poxviruses, are often not amenable to X-ray crystallography, nuclear magnetic resonance (NMR) analyses, or fine scale reconstruction by cryo-electron microscopy. Large scale structural details regarding the architecture of the vaccinia virion, the largest human tropic virus (with dimensions of air-dried virion of ~ 330 nm x 260 nm x 125 nm)1, and one of the
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most intensively studied poxviruses, have been predominantly derived from conventional and cryo-electron microscopy analyses.2,3,4,5,6,7,8 While these studies have produced a wealth of information regarding the architecture of vaccinia, few structural features remain unambiguous; these are limited to the presence of a proteinaceous core containing the genome and viral enzymes, as well as the presence of proteinaceous lateral bodies, surface tubular elements and an exterior membrane.9 Due to the lack of a well-resolved model for vaccinia the application of additional physical measurement techniques is warranted. Secondary ion mass spectrometry (SIMS) is an analytical technique that may provide additional insight into the structure of small biological particles such as the vaccinia virion.10 For SIMS, a sample is bombarded by a ‘primary’ ion beam with sufficient kinetic energy to implant into a solid sample and eject (‘sputter’) atoms, molecules and molecular fragments, resulting in sample surface erosion.11 For magnetic sector SIMS, reactive primary ion species are typically used to enhance the yield of ions generated from the sample (‘secondary ions’), such as a Cs+ primary beam to enhance the yield of negative secondary ions. At the initiation of sputtering the conditions are non-equilibrium because the concentration of the primary ion species at the implantation depth in the sample (10s of nm) is building up and causing the yield of secondary ions (ions generated per atoms sputtered) at the eroding surface to change. Equilibrium conditions are reached when the concentration of the primary ion species in the eroding surface is quasi-constant because the primary ions are ejected at approximately the same rate as they are implanted into the surface. The major issue in the current study is that non-equilibrium conditions are not well characterized, and they are expected to persist to a depth of 10s of nanometers11, which represents a substantial portion of nanometer-scale particles such as vaccinia virions. For this study, we use the Cameca NanoSIMS 50, which is a magnetic sector SIMS instrument that provides unprecedented sensitivity and mass specificity at high spatial resolution.10,12 This approach to SIMS relies on rare stable isotopes incorporated in molecules of interest to enable localization.12 Biological imaging with isotopic labeling has previously been implemented to analyze sub-cellular structures in eukaryotic systems.10,13 In addition, 3D quantitative depth resolved imaging has been utilized to examine individual cells,14 as well as the elemental composition in individual 1-2 micron sized bacterial endospores.15 Depth profiling small biological structures has the potential advantage of higher spatial resolution and sensitivity than can be achieve laterally.15 However, due to the relatively large size of the cells analyzed in the aforementioned studies, the rate of sample surface erosion during non-equilibrium sputtering conditions was neglected when constructing depth profiles. Here we present a series of experiments to characterize virion sputtering. We first present SIMS depth profile data for vaccinia virions with isotopically labeled DNA to demonstrate the problem. Vaccinia virions were labeled with 15N-labeled thymidine by providing it in the medium for the cells employed for virus growth. The labeled virions
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Analytical Chemistry
were extracted and purified for analysis. SIMS imaging depth profiling was employed to generate the raw depth distribution of 15N/14N ratio; measuring a ratio controls for changes in ion yield with depth. To ensure the 15N/14N data related specifically to the virion DNA distribution, we tested the assumption that the 15N-thymidine was exclusively incorporated into the DNA by fractionating the 15N-labeled virions into nucleic acids, proteins and lipids and analyzing them by SIMS for 15N/14N ratio. Then we present data from correlated SIMS sputtering and atomic force microscopy (AFM) dimensional analysis to develop a non-linear, non-equilibrium sputter rate model. The majority of the data are for a Cs+ primary beam, including data for glutaraldehyde-fixed virions, but we also show results for O- and Ga+ beams. Finally, we use our non-linear model to correct the depth profiles for the 15N-DNA virions, and the results are compared to the standard model for vaccinia virion architecture. EXPERIMENTAL SECTION Virus Samples. The vaccinia virus was grown in BSC-40 cells, an African green monkey kidney cell line. Cells were maintained at 37oC in 5% CO2 in the presence of Dulbecco’s Modified Eagle’s medium (Life Technologies, cat 12100-061) containing 10% fetal bovine serum, 0.12 mg/ml penicillin (Sigma), 0.2 mg/ml streptomycin (Sigma) and 250 mg/l fungizone. Vaccinia virus strain WR was maintained as previously described.16 Infected cells were harvested by centrifugation at 3,000 x g at 4oC, re-suspended in 3 ml of 10 mM Tris-HCl, pH 8.0, and frozen at -80oC. All further steps were carried out at 4oC in a buffer containing 10 mM Tris HCl, pH 8.0. The cell suspension was thawed, then disrupted by Dounce homogenization and the nuclear fraction was separated by centrifugation at 900 x g for 10 min. The post nuclear supernatant was saved, and the nuclear pellet was resuspended in 3 ml, re-extracted by Dounce homogenization and centrifuged as described above. The supernatant was removed, combined with the previous supernatant, layered on a 6-ml cushion of 36% sucrose, and centrifuged in a Beckman SW41 rotor at 19,000 rpm for 80 min. The pellet was re-suspended in 1 ml, sonicated 3 times for 30 seconds each time, layered on a 10.5 ml 24–40% sucrose gradient, and centrifuged in an SW41 rotor at 14,000 rpm for 40 min. A visible band in the middle of the tube was collected in a volume of approximately 4 ml, diluted to 12 ml and centrifuged in an SW41 rotor at 16,000 rpm for 60 min. Virus pellets were re-suspended in 300 µl of deionized water and virus concentrations were determined by measuring light scattering at OD260 nm; concentrations ranged from approximately 5 – 15 OD corresponding to 6.0x1010 - 1.8x1011 particles/ml, respectively. Finally, the virus pellet was washed 3 times by re-suspending the pellet in 750 µl of deionized water, centrifuging at 14,000 x g for 10 minutes in an Eppendorf microfuge and finally re-suspending in 300 µl of water. The chemical fixation procedure is in the Supporting Information (SI).
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Labeling. To prepare 15N-labeled virus, six 150 mm confluent monolayers of BSC-40 cells were infected with virus at a multiplicity of infection equal to 1 and incubated in medium supplemented with 30 ug/ml 15N-labeled thymidine (Cambridge Isotopes, cat NLM-3901-0) for 48 h at which time complete cytopathic effect was observed and virus isolation was performed as described above. Molecular fractionation was carried out to verify that only the DNA was labeled (see SI). 15N-Thymidine
Atomic Force Microscopy. The goal of sample preparation for AFM and SIMS was to disperse virons on a smooth, conducting substrate (Si metal) with sufficient density that multiple single virions could be analyzed within 10 µm x 10 µm regions. To this end, 1 µl droplets of the original or diluted vaccinia virus suspension were deposited onto 5 mm x 5 mm silicon substrates (Ted Pella, Inc., Redding, CA), allowed to settle for ~10 minutes, and then rinsed with double-distilled water and dried with a gentle stream of nitrogen gas. The AFM set up includes an optical microscope with a CCD camera, which we used to assess the density of virions on the substrate and identify areas with low density of virions for further AFM and SIMS correlated analysis (Fig. S1). AFM imaging allowed the identification of regions with dispersed single virions located close to fiducial markings (Fig. S1). AFM images were collected with a Multimode Nanoscope IV atomic force microscope (Bruker Corporation, Santa Barbara, CA.) operated in tapping mode. For high-resolution imaging of viral structures, SuperSharpSilicon (SSS) AFM probes (NanoWorld Inc., Neuchatel, Switzerland) with a force constant of 10 – 130 N/m (tapping frequencies of 200 – 500 kHz) were utilized. For large-scale scans, greater than approximately 20 µm2, dimension microactuated silicon AFM probes (DMASP, Bruker Corporation, Santa Barbara, CA) with a force constant of 1-5 N/m (tapping frequencies of 150-250 kHz) were utilized. Phase and height images were collected simultaneously. Height data corresponds to the change in piezo height needed to keep the oscillation amplitude of the AFM cantilever constant. Phase data correspond to surface-induced changes in phase of the AFM cantilever oscillation. Height images allow quantitative height determinations, providing precise measurements of virion’s heights and surface topography. Heights of virions were measured from height images using the AFM off-line section command, which allows measurements of vertical distance (height) and horizontal distance. Phase images do not provide height information; however, they can display more structural detail and contrast compared with height images and therefore they are often used for presentation purposes. Other AFM experimental procedures and operating parameters were as previously described.1 After AFM imaging, the sample was subsequently transferred to the NanoSIMS and the target regions were located with optical and secondary electron imaging (Fig. S2). Further details regarding this procedure can be found in the SI. Secondary Ion Mass Spectrometry Analysis. SIMS analyses of regions of dispersed, single virions identified by AFM were performed using the LLNL NanoSIMS 50 (CAMECA
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Analytical Chemistry
Instruments, Gennevilliers, France). Only virions oriented with the minor axis perpendicular to the substrate (i.e., broad side up) were analyzed (Fig. S3). These virions were analyzed without metal coating for this study because we found that SIMS analysis of sputter coated virions resulted in irregular surface structures (Fig. S4). Samples were evacuated to ultra-high-vacuum (UHV) conditions prior to analysis (
