Haloarchaeal host strains and viruses and growth conditions

Haloarchaeal pleomorphic viruses HRPV-2 and HRPV-6 as well as their host strains Halorubrum sp. SS5-4 and SS7-4 were initially isolated from samples collected in a solar saltern samples in Samuth Sakhon, Thailand21,22 and the non-pigmented derivative of SS7-4 (see below), respectively, were used in this study. Halorubrum sp. PV65 was initially isolated from samples collected in a solar saltern in Trapani, Sicily, and used as a non-host strain. Haloarchaeal strains were grown aerobically in Modified Growth Media23 (MGM) at 37 °C. MGM broth, plates and soft-agar contained 23, 20 and 18% (wt v−1) artificial salt water (SW, HaloHandbook [http://www.haloarchaea.com/resources/halohandbook/Halohandbook_2008_v7.pdf]) respectively. Viruses were propagated on lawns of host cells grown in MGM soft agar. HRPV-2 and HRPV-6 storage solutions were prepared using the top agar of semi-confluent plates and incubating it together with MGM (2 ml for each plate used) at 37 °C with shaking for 2 h. The debris was removed by centrifugation (Sorvall F12, 11,000 × g for 40 min at 4 °C) and the cleared lysate was stored at 4 °C. 12% SW-HEPES was diluted from the 30% SW-HEPES stock solution in which Tris-HCl buffer (pH 7.2) was replaced by HEPES buffer of the same concentration and pH.

Production of non-pigmented mutant strain

Ethyl methane sulphonate (EMS) was used to mutate cells of Halorubrum sp. SS7-4 and screen for non-pigmented cells conducted as described by Mevarech and Werczberger24. Briefly, cells were grown to exponential growth phase and collected by centrifugation (Sorvall SA600, 9700 × g at ambient temperature) followed by resuspension in an equal volume of buffer containing 50 mM Tris-HCl pH 7.2 and 3 M NaCl. EMS (Sigma M0880) was added to the final concentration of 100 µg ml−1. Cells were incubated with EMS at 37 °C with aeration for 2 h after which cells were washed twice with the buffer. Finally cells were resuspended in equal volume of MGM broth and grown until they reached the density of ~1 × 109 cfu ml−1. Cell suspension was diluted and plated on MGM media plates and grown at 37 °C until colonies appeared.

Purification of viruses and spike proteins

HRPV-6 and HRPV-2 virions were purified from the storage solutions first by concentrating them with 10% polyethylene glycol 6000 (PEG6000) at 4 °C for one hour with gentle stirring. Precipitated viral particles were collected by centrifugation (Sorvall F12, 11,000 × g at 4 °C) and viral pellets were resuspended in 18% SW. Aggregates were removed by centrifugation (Sorvall F14, 11,500 × g at 4 °C). To obtain 1× purified virus, concentrated viral solution was subjected to rate-zonal centrifugation (104,000 × g at 15 °C for 2 h for HRPV-6 and for 3 h 30 min for HRPV-2, Sorvall AH629) in linear 5 to 20% (wt vol−1) sucrose gradient. The 1 × purified virus was further purified with a CsCl gradient (ρ=1.3 g ml−1) by equilibrium centrifugation (79,000 × g for 20 h at 15 °C, Sorvall AH629) to obtain 2 × -purified material that was concentrated by centrifugation (114,000 × g for 3 h at 15 °C, Sorvall T647.5). For crystallization of the spike proteins, two methods were used to dissociate and purify the proteins directly from highly purified virions (2×-purified virus). For HRPV-2 VP5 protein, highly purified virions (200 µg ml−1) were dissociated with Nonidet P40 at a final concentration of 0.1% (v v−1) in HRPV-buffer containing 20 mM Tris-HCl pH 7.5, 1.5 M NaCl, 100 mM MgCl 2 and 2 mM CaCl 2 . Soluble VP5 proteins were separated from the other dissociation products by rate zonal centrifugation (Sorvall TH641, 210,000 × g, 4 h at 15 °C) in linear 5 to 20% sucrose gradient in the HRPV-buffer. Soluble VP5 proteins were collected, concentrated and washed with detergent removal buffer (20 mM Tris-HCl pH 7.5, 0.5 M NaCl) using ultrafiltration (Amicon Ultra Centrifugal Filter Devices, Millipore, 10,000 nominal molecular weight limit). Detergent was removed from the sample using Detergent Removal Spin Columns (Pierce) as instructed by the manufacturer. Purified sample was washed with detergent removal buffer and concentrated by ultrafiltration as described above.

The ectodomain of the HRPV-6 VP5 were released from highly purified virions (2×virus, 200 µg ml−1) using proteinase K (Fermentas) digestion at a final concentration of 20 µg ml−1 in 12% artificial salt water (SW). After the digestion (3 h, 37 °C) soluble ectodomains of the spike protein were separated from the other dissociation products by rate zonal centrifugation (Sorvall AH629,112,400 × g, 7 h at 15 °C) in a linear 5–20% sucrose gradient in 12% SW. Soluble proteins were collected and concentrated by ultrafiltration (Amicon Ultra Centrifugal Filter Devices, Millipore, 30,000 nominal molecular weight limit) and washed with gel filtration buffer (20 mM Tris-HCl pH 7.2, 1 M NaCl, 40 mM MgCl 2 ). The ectodomains of the spike protein was separated from the remaining proteinase K by gel filtration (Superdex 200, HiLoad 16/60, GE Healthcare) and fractions containing the spike protein devoid of proteinase K were pooled, concentrated and washed by ultrafiltration (Amicon Ultra Centrifugal Filter Devices, Millipore, 100,000 nominal molecular weight limit). The buffer of the final protein sample contained 20 mM Tris-HCl pH 7.2, 1.6 M NaCl and 10 mM MgCl 2 .

Labelling of HRPV-6

Highly purified (2×-purified) HRPV-6 virions produced in the non-pigmented SS7-4 host were labelled using R18 (O-246, Molecular Probes). Approximately 1.7 × 1013 plaque forming units per millilitre (PFU ml−1) were mixed well with 45 µg ml−1 (62 µM) of the lipophilic dye and divided in two aliquots of equal volume. After one hour-incubation at 37 °C in dark, proteinase K (Fermentas) was added to one aliquot at final concentration of 200 µg ml−1 and both aliquots were incubated further for another 1 h at 37 °C. Virions were purified by rate zonal centrifugation in a linear 5–20% sucrose gradient (Sorvall TH641, 154,000 × g for 3 h 30 min at 15 °C). Light scattering zones containing the virions were collected, concentrated and washed by ultrafiltration (Amicon Ultra Centrifugal Filter Devices, Millipore, 100,000 nominal molecular weight limit). The infectivity of the labelled particles was determined by titration and the incorporation of fluorescent label by measuring the released fluorescence in counts per second (cps) at Ex 560 nm and Em 590 nm in the presence of 0.17% Triton X-100 (v v−1) in 12% SW-HEPES buffer using FluoroMax-4 spectrofluorometer (HORIBA Jobin Yvon Inc). Specific infectivity was determined as plaque forming units per counts of fluorescence in one second (PFU cps-1) and it was 72 PFU cps−1 for the wild-type HRPV-6 particles and 8.0 × 10−2 PFU cps−1 for the spikeless particles. Analysis of the viral particles in SDS-PAGE showed that most of the spike protein was removed by the proteinase K digestion (Supplementary Figure 4).

Adsorption tests

The host culture to be tested was diluted to A 550nm of 0.1 and grown to an approximate cell density of 2–3 × 108 colony-forming units per millilitre (CFU ml−1, A 550nm 0.45–0.5) at 37 °C with shaking (~16 h). Cells were collected by centrifugation (Sorvall SA600, 9700 × g, 5 min, RT) and resuspended in either MGM or 12% SW- HEPES. Viruses were added to the cell culture at multiplicity of infection (M.O.I.) of 6 × 10−7. Samples of 200 µl were withdrawn at indicated time points, cells were collected by centrifugation (Eppendorf Centrifuge 5415D 9300 × g, 5 min, RT) and the supernatant and the cell pellet, resuspended in 200 µl of fresh growth medium, were titrated in 100 µl aliquots with host cells as described before5.

Fluorescence microscopy

Non-pigmented haloarchaeal SS7-4 host cells produced in this study (see Production of non-pigmented mutant strain) were grown as described for the adsorption test. To a solution of cells at their logarithmic growth phase (A 550nm of ~0.4–0.5, ~1–2 × 108 CFU ml−1) labelled viruses were added at an approximate M.O.I. of 10–50 (virions devoid of proteinase K treatment) and approximately the same number of proteinase K-treated viral particles as determined by released fluorescence. Suspensions were incubated at 37 °C for 15 min with shaking after which cells from 1 ml of sample were collected by centrifugation (Eppendorf Centrifuge 5415D, 5900 × g, 5 min, RT) and washed once with MGM broth. Cells were incubated for additional 10 min after which the media was changed into 12% SW-HEPES by pelleting and resuspension of the cells twice.

For microscopy, cells in 12% SW-HEPES were fixed with paraformaldehyde (final concentration 5.2% v v−1) for 20 min and washed with 12% SW-HEPES three times. Aliquots of fixed cell suspension were mounted on 1% agarose in 12% SW-HEPES and visualised using a Leica DM6000B (Wezlar, Germany) microscope with ×63/1.40 HCX PL APO Lbd.bl. oil objective with ×1.25 magnification changer. Final pixel size is 69 nm. Differential interference contrasting (DIC) was used with bright field and Semrock Brightline TRITC-B (ex 543/22, em 593/40) filter to detect the fluorescence of R18. A Hamamatsu Orca Flash 4.0v2 camera was used to capture 16 bit images. 700 ms exposure time was used for fluorescence channel Leica Application Suite X (LAS X) and Fiji ImageJ 1.51n was used for image processing. The fluorescence signal of all obtained images were adjusted to maximal fluorescence of 1200 and within one experiment, images obtained from DIC and fluorescent channels were all aligned manually in the same way.

Virus-cell and virus-liposome fusion assay

Virus fusion with target membranes was monitored by fluorescence dequenching of R18-labelled virions by standard techniques25. To this end, purified (1×-purified) HRPV-6 virions produced in the non-pigmented Halorubrum sp. SS7-4 host were labelled with R18 at a self-quenching concentration as described above. R18 labelled HRPV-6 virions were separated from excess probe by using a sephadex G-75 column (GE Healthcare). As a negative control, R18 labelled and purified HRPV-6 particles were treated with proteinase K at 200 μg ml−1 for 1 h at 37 °C to eliminate any exposed membrane proteins.

In the case of virus–cell fusion, non-pigmented haloarchaeal cells were used at their logarithmic growth phase (OD 0.4–0.5, adsorption at 550 nm). For virus–liposome fusion, total lipids were extracted from non-pigmented haloarchaeal SS7-4 host cells based on one phase alcoholic solvent system based on methanol-chloroform-water (1:2:0.8 v v‒1) extraction as described before26 and resuspended in HRPV buffer. Fresh liposomes were prepared by the freeze-thaw and extrusion method27 using a polycarbonate filter with a pore size of 0.2 mm (Avanti Polar Lipids).

To monitor fusion, R18 labelled HRPV-6 virions (~108 PFU) were mixed with either non-pigmented haloarchaeal cells at a MOI of 10–20 or with liposomes derived from SS7-4 host cells (5 mg ml‒1) in a fluorimeter cuvette under continuous stirring at the indicated temperatures. Fluorescence dequenching was recorded continuously every 60 s at 585 nm at an excitation wavelength of 565 nm using a fluorescence spectrophotometer (Varian Eclypse, Agilent Technologies) with 5 and 10-nm slit width for excitation and emission, respectably. The base value at time 0 was defined as 0% lipid mixing and the maximal extent of R18 dilution was determined by the addition of Triton X-100 (final concentration 0.1%) after the lipid mixing of each condition had concluded. After data collection, the lipid mixing kinetic was fitted to a single exponential fit using Eq. (1).

$${\mathrm{Lipid}}\,{\mathrm{mixing}}\,\left( {t} \right){\mathrm{ = Lipid}}\,{\mathrm{mixing}}_{{\mathrm{max}}} \times {\mathrm{(1 - exp( - \tau }} \times {t}{\mathrm{))}}$$ (1)

where τ is the time constant and Lipid mixing max corresponds to the maximum lipid mixing value at infinitive time.

LC-MS/MS

Peptides were quenched with 10% trifluoroacetic acid (TFA) and purified with C18 microspin columns (Harvard Apparatus, USA) eluting the samples to 0.1% TFA in 50% acetonitrile (ACN). The dried peptides were reconstituted in 30 µl 0.1% TFA in 1% ACN (buffer A). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was carried out on an EASY-nLC 1000 (Thermo Fisher Scientific, Germany) connected to a Velos Pro-Orbitrap Elite/Q Exactive hybrid mass spectrometer (Thermo Fisher Scientific, Germany) with nano electrospray ion source (Thermo Fisher Scientific, Germany). The LC-MS/MS samples were separated using a two-column setup consisting of a 2 cm C18 Pepmap column (#164946 Thermo Fisher Scientific, Germany), followed by 15 cm C18 Pepmap analytical column (#164940 Thermo Fisher Scientific, Germany). The linear separation gradient consisted of 5% buffer B in 5 min, 35% buffer B in 60 min, 80% buffer B in 5 min and 100% buffer B in 10 min at a flow rate of 0.3 µl min−1 (buffer A: 0.1% TFA in 1% acetonitrile; buffer B: 0.1% TFA acid in 98% acetonitrile). 4 µl of sample was injected per LC-MS/MS run and analysed. Full MS scan was acquired with a resolution of 60,000 at normal mass range in the orbitrap analyzer the method was set to fragment the 20/10 (QE 10 and OQ top 20) most intense precursor ions with CID (Elite OE/HCD with QE) (energy 35). Data were acquired using LTQ Tune software.

Acquired MS2 scans were searched against home-made protein database using the Sequest search algorithms in Thermo Proteome Discoverer. Allowed mass error for the precursor ions was 15 ppm. And for the fragment in 0.8 Da/or 0.05 Da(Q Exactive). A static residue modification parameter was set for carbamidomethyl +57,021 Da (C) of cysteine residue. Methionine oxidation was set as dynamic modification +15,995 Da (M). The analysis was carried out at the Protein Chemistry Core Laboratory, Institute of Biotechnology, Helsinki Institute of Life Science HiLIFE, University of Helsinki.

Crystallization and data collection

Both HRPV-2 VP5 and HRPV-6 VP5 proteins were crystallized in 96-well plates (Greiner Bio-One Ltd, Stonehouse, England) using the sitting-drop vapour-diffusion method at 21 °C with a Cartesian Technologies MIC4000 robot28,29 (Digilab). HRPV-2 VP5 crystals grew in a solution containing 12% (w v−1) PEG 4000, 0.1 M Na-HEPES pH 7.5 and 0.1 M NaCl from the MemSys crystallization screen (Molecular Dimensions). Crystals were first stabilised in the same reservoir solution but containing 30% (w v−1) PEG 4K and then dipped into oil (Perfluoropolyether PFO-X175/08, Hampton Research) before flash-cooling in liquid nitrogen. A dataset (space group P2 1 2 1 2 1 ) was collected on beamline I24 at Diamond Light Source (DLS, Didcot, England) to 2.5 Å resolution (unit cell a = 48.1, b = 93.2, c = 121.8 Å, α = β = γ = 90°). Initial crystals of HRPV-6 VP5 grew in 1.6 M Ammonium Sulphate, 0.1 M Citrate pH 5.0 from the Ammonium Sulphate Grid Screen (Hampton Research) and were further optimized by adjusting the pH between 5.4 and 5.8 and the precipitant between 1.6 and 1.8 M. Due to the high NaCl concentration in the protein buffer, NaCl was added to all the reservoir solutions to a final concentration of 1.6 M. For phasing purposes, HRPV-6 VP5 crystals were briefly immersed in the reservoir solution containing 0.2 M NaBr and 30% (w v−1) ethylene glycol prior to flash-cooling in liquid nitrogen. In order to optimize the anomalous signal from the bromine, a dataset was collected close to the bromine edge at 0.9150 Å on the I02 beamline at DLS employing the inverse-beam method. Crystals diffracted to 2.7 Å resolution and belonged to space group P6 5 22 with unit cell parameters a = b = 114.3, c = 445.2 Å, α = β = 90°, γ = 120°. HRPV-2 and HRPV-6 VP5 datasets were indexed, integrated, and scaled with XIA230,31,32.

Structure determination and refinement

Structure determination of HRPV-6 VP5 via the single-wavelength anomalous dispersion (SAD) method used the anomalous scattering from the bromine atoms. HKL2MAP33,34 determined the position of 20 bromine atoms, which were then used in PHENIX.AUTOSOL35 for phasing. The experimental electron density map was clearly interpretable and the structure was initially partially built with PHENIX.AUTOBUILD35 and then completed by manual building in COOT36. Two molecules were present in the asymmetric unit, although no density could be seen for half of one of them. HRPV-2 VP5 (62% sequence identity to HRPV-6 VP5) was solved by molecular replacement using PHASER37 using HRPV-6 VP5 as a search model. Both structures were refined with AUTOBUSTER38 to R work and R free of 21.8/23.3% for HRPV-6 VP5, and 22.4/24.1% for HRPV-2 VP5. Final structures were validated with MOLPROBITY39 with more than 97% of residues in the Ramachandran favoured regions and no outliers. Data collection and refinement details are presented in Table 1, and a stereo view of the final electron density maps are shown in Supplementary Figure 5.

Protein in silico analyses

The hydrophobic regions of HRPV-2 and HRPV-6 VP5 proteins were predicted using TMpred server9, Phobius40 and MPEx41.

Sample preparation for electron microscopy

VP5 spikes on HRPV-6 are very sensitive to ionic strength and only stable in 1.6 M NaCl or above. At lower concentrations of NaCl the spikes fall apart. As a result a careful optimisation of NaCl concentration and incubation time was performed. For cryo-EM, samples were prepared by diluting the purified virus (10×) in the buffer containing 250 mM NaCl immediately prior vitrification. Aliquots of 4 μl were added onto a glow-discharged holey carbon copper grid (C-flat, CF-2/1-2C; Protochips). Grids were blotted for 3 s, in 90% relative humidity, and vitrified in liquid ethane with a plunger device (Vitrobot; FEI). In order to keep the spikes intact and in native form, grids were plunged within 30 s of dilution to low salt buffer. To assess the salt concentration effect on the virions and spike, HRPV-6 in 1.5 M NaCl buffer were plunge-frozen on the grids as control.

To study heat-treated particles of HRPV-6, 100 µl of highly purified particles (~1 × 1012 pfu ml−1) were split into two 50 µl aliquots. One aliquot was incubated at 55 °C and one at room temperature for 30 min in HRPV-buffer. Aliquots of 3 µl were added onto glow-discharged copper quantifoil grids (R1.2/1.3), blotted for 4.5 s and vitrified in liquid ethane with a plunger device (Vitrification robot; Leica).

Cryo-electron microscopy data collection

Cryo-EM data were collected at the national Electron Bio-Imaging Center (eBIC), at the Diamond Light Source on a FEI Titan Krios transmission electron microscope operating at 300 kV. The Krios was equipped with an energy filter (GIF Quantum, Gatan) operating in zero-loss mode with a 20 eV slit and a Volta phase plate (VPP; Thermo Fisher). Single axis tilt series (from ‒45° to +45° with angular increments of 3°) were collected using EPU software (FEI) on a direct electron camera (K2 Summit, Gatan). At each tilt, images were recorded as a movie consisting of eight frames with a total exposure of 1.6 s per tilt and at a calibrated magnification of ×22,222 in a single electron counting mode, corresponding to a pixel size of 2.25 Å. The datasets were collected in focus and the standard autofocusing routine implemented in EPU was used at every tilt angle. Cumulative electron dose was kept constant in all datasets and the irradiated area on the VPP was changed after each tomogram. Images of heat treated particles were collected at the University of Helsinki cryo-EM facility on a Talos Arctica electron microscope operating at 200 kV and using Falcon III detector (Thermo Fisher).

Image pre-processing and tomogram generation

Drift correction42 was used to correct for the electron beam induced motion by averaging eight frames for each tilt. 25 tilt series were aligned by patch tracking in IMOD package43. The six best tilt series, which showed consistent contrast, were reconstructed into tomograms with a final pixel size of 2.25 Å. Altogether 247 HRPV-6 virions were picked in Bshow44 and extracted from the tomograms using Jsubtomo45 for further analysis.

Sub-tomogram averaging

For initial template generation, extracted virion volumes were low pass filtered to 80 Å using Bsoft44. Spikes (8953 in total) were manually picked from the volumes of HRPV-6 using Dynamo46. All picked spikes were extracted from the unfiltered tomograms into boxes of 128 × 128 × 128 voxels. Sub-tomogram averaging was carried out in Dynamo46, following protocols we have established earlier45,47. In the first stage of refinement, both the locations and directions of the picked particles were allowed to change. The angle around the spike long axis (azimuth) was kept fixed. The refinement was carried out while restricting the resolution to 36 Å, during which a large spherical mask (radius 63 pixels) and full cylindrical symmetrization were applied to roughly align the spikes.

For the second stage, a customized post-processing plugin47 was designed to carry out gold-standard refinements where only the azimuth angle was refined. The dataset was randomly split into two datasets consisting of 4502 and 4451 particles and each dataset was averaged to produce a template for refinement. Independent refinements were carried out on the two datasets. At the end of each iteration, the Fourier shell correlation (FSC) between the two averages was computed and the resolution estimated using a criterion of 0.143. Two shells in Fourier space were subtracted from the estimated resolution value, and the difference was used as the threshold for a low-pass filter for the next iteration. A spherical mask (radius 32 pixel), encompassing only the spike ectodomain was used, and no symmetry was assumed. After this stage, 1436 particles were removed from the dataset due to overlaps. Averaging the remaining particles produced an ellipsoidal-shaped spike density, which was connected with the membrane by one, possibly two small stalks.

In the third stage, an ellipsoidal mask (semi-principal axes of length 40, 24 and 24 pixels in the x, y and z direction), encompassing only the spike ectodomain was used. In this stage, the azimuth angle was further refined with small angular search steps. After this stage, 629 particles were removed from the dataset due to low cross-correlation. The coordinates of the remaining particles were plotted and 226 particles located obviously outside of the virus were further removed.

In the fourth stage, all six parameters (three location coordinates and three Euler angles) were allowed to change simultaneously. After this stage, 741 particles were removed due to low cross-correlation or overlap, giving finally 3112 and 2945 particles for the two datasets. The final map resolution of 16 Å was estimated by FSC, using 0.143 as threshold. To assess the salt concentration effect on the virions and spike, 1200 spikes from HRPV-6 in 1.5 M NaCl buffer were cylindrically aligned and averaged.

X-ray structure fitting

X-ray structure of HRPV-6 VP5 was fitted as a rigid body into the segmented EM density of the VP5 spike in UCSF Chimera48. Initial manual fitting that placed the C-terminus proximal to the membrane was improved by ‘fit in map’ function (correlation 0.83).

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.