www.sciencemag.org/cgi/content/full/science.aaf9620/DC1

Supplementary Materials for

An atomic model of HIV-1 capsid-SP1 reveals structures regulating assembly and maturation

Florian K. M. Schur, Martin Obr, Wim J. H. Hagen, William Wan, Arjen J. Jakobi, Joanna M. Kirkpatrick, Carsten Sachse, Hans-Georg Kräusslich, John A. G. Briggs*

*Corresponding author. Email: john.briggs@embl.de

Published 14 July 2016 on Science First Release

DOI: 10.1126/science.aaf9620

This PDF file includes:

Materials and Methods Figs. S1 to S5 Tables S1 to S2 Full Reference List Captions for Movies S1 and S2

Other Supplementary Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.aaf9620/DC1)

Movies S1 and S2

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Materials and Methods

HIV-1 purification and Virus-like particle assembly

HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS; Biochrom), penicillin (100 IU/mL), streptomycin (100 µg/mL) and 4 mM glutamine. For virus production, cells were transfected with the proviral plasmid pNL4-3 (PR-) using calcium phosphate following standard procedures. At 44 h post transfection, tissue culture supernatant was harvested and filtered through 0.45 µm nitrocellulose filters. Virus was enriched by ultracentrifugation through a 20% (w/w) sucrose cushion and further purified by centrifugation through an iodixanol density gradient (34). Concentrated virus samples were resuspended in phosphate buffered saline (PBS), treated with 1% paraformaldehyde for 1 h on ice and stored in aliquots at -80°C. Purity of samples and Gag maturation state were verified by SDS-PAGE followed by silver staining and immunoblotting.

ΔMACANCSP2 protein was expressed in E. coli cells and purified as described previously (11). In brief, the cell lysate was precipitated by ammonium sulphate (25% saturation), and further purified using a combination of anion and cation exchange chromatography. Homogenously pure ΔMACANCSP2 was transferred into a storage buffer (50 mM Hepes, pH 7.5, 500 mM NaCl, 10% Glycerol, 10 µM ZnCl2) and concentrated to 4 mg/ml.

In order to assemble ΔMACANCSP2 VLPs, protein stock was dialyzed against assembly buffer (50 mM Hepes, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM TCEP) in the presence of nucleic acid (single-stranded DNA 73mer oligonucleotide, 1:10 molar ratio oligonucleotide:protein) for 16h at 4°C. In vitro assembled particles were harvested by centrifugation (10 000g, 5 min) and resuspended in fresh assembly buffer. Additionally, particles for the binding analysis of the maturation inhibitor BVM were assembled in presence of the compound (100 µg/ml), and washed in 20% methanol prior to resuspension. Inhibition of CA-SP1 cleavage was validated by incubating 500 nM ΔMACANCSP2 VLPs, assembled in the presence or absence of 100µg/ml BVM, with 100 nM recombinant HIV-1 PR in PR assay buffer (50 mM MES, pH 6.5, 150 mM NaCl, 1 mM EDTA, 1mM TCEP, 1mg/ml BSA) for 1h at 25°C. Semi-quantitative western blots were performed with polyclonal sheep antiserum against CA and secondary antibodies coupled to Alexa fluorescent dyes for detection with an Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE). Band intensities were quantitated using ImageJ to determine relative accumulation of CA-SP1 cleavage products (Fig. S5).

Approximate quantification of the amount of BVM incorporated into VLPs was performed by UPLC-MS-MS (Waters Q-Tof Premier Mass Spectrometer). Protein and nucleic acid from the BVM containing sample was denatured by a methanol-acetonitrile mixture (80:20) and separated by retention in a centrifugal filter unit (molecular weight cut-off 30 kDa). The flow-through was collected, dried in a SpeedVac concentrator at 37°C and reconstituted in UPLC sample buffer (20 mM ammonium formate, pH 8.0, 20% acetonitrile, 5% methanol, 1% DMSO). The reconstituted sample was applied to a

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C18 reverse phase column and eluted by a linear gradient of 20 – 95% acetonitrile in 20 mM ammonium formate, pH 8.0. The eluate was analyzed by electrospray ionization in negative ion mode with tandem MS/MS detection (Q-TOF). The integral area of the 583.40 Da BVM monoisotopic peak was used for quantification, compared to a calibration curve prepared by mixing BVM at known concentrations with pre-assembled ΔMACANC VLPs. In data from four independent experiments we observed a molar ratio of 2.6 BVM : 6 ΔMACANCSP2 (range 0.9-4.1) but this may partly reflect non-specific binding.

Cryo-electron tomography

Degassed 2/1-3C or 2/2-3C C-flat grids were glow discharged for 30 seconds at 20 mA. VLP or virus solution was diluted with 10nm colloid gold in either PBS or VLP sample buffer. Then 2 µl of the solution was applied to grids and plunge frozen in liquid ethane. Grids were stored under liquid nitrogen conditions until imaging.

Data acquisition and image processing was performed identically for all three datasets (untreated ΔMACANCSP2, ΔMACANCSP2+BVM and immature HIV-1 (D25A) virus particles) unless otherwise stated (see also Table S1).

All imaging was done on a FEI Titan Krios transmission electron microscope operated at 300 keV, through a Gatan Quantum 967 LS energy filter using a slit width of 20 eV, onto a Gatan K2xp direct detector using SerialEM software (35). For navigation and search purposes, low-magnification montages were acquired. Prior to data acquisition, a full K2 gain reference was acquired and the Quantum energy filter was fully tuned. FEI AutoCTF software (part of the FEI Volta phase plate package) (36) was used for microscope tuning. The nominal magnification for data collection was 105,000x, giving a calibrated 4K pixel size of 1.35 Å. The tilt range was from 0 to 60° and -60° in 3° steps, in a newly-developed dose-symetric tilt scheme (0, +3, -3, -6, +6, +9, -9, -12, etc.), the implementation of which is described in (12). Tilt images were acquired as 8K x 8K super-resolution movies of 6-10 frames with a set dose rate of 1.5-8 e/Å2/sec. Tilt series were collected at a range of nominal defoci between -1.5 and -5.0 µm and a target total dose of 90 to 150 e/Å2.

Image processing

K2Align software, which uses the MotionCorr algorithm (14), was used to align frames and Fourier crop the aligned images to 4K x 4K, minimizing aliasing effects. Defocus determination was performed using CTFFIND4 (37) or by fitting theoretical CTF-curves to radially-averaged powerspectra using MATLAB (MathWorks) scripts as described previously (38). CTF-estimation was done for each tilt individually. Each image was low-pass filtered according to the cumulative electron dose (exposure filtering). Exposure filters were calculated using the exposure-dependent amplitude attenuation function and critical exposure constants as previously determined (15). For a small number of tilt-series with higher cumulative electron dose, high-tilts were removed prior to further

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processing.

Exposure-filtered images were then used for tomogram reconstruction in the IMOD software package (39). CTF-correction was performed by the “ctfphaseflip”-program implemented in IMOD (40). CTF-corrected unbinned tomograms were reconstructed and subsequently 2x (2.7 Å/px), 4x (8.1 Å/px) and 8x (10.8 Å/px) binned using anti-aliasing. VLPs and virus particles were identified in the 8x-binned tomograms using the Amira visualization software (FEI Visualization Sciences group) and for each particle the center and radius was determined using the electron microscopy toolbox (41). Tomograms for untreated ΔMACANCSP2, ΔMACANCSP2+BVM and protease defective HIV-1 (D25A) contained 285 VLPs, 383 VLPs and 484 viruses, respectively. All three datasets were processed independently.

Sub-volumes with a size of (389)3 Å were extracted from 8x binned tomograms on the surface of each particle according to the determined radius. Subtomogram averaging was performed using scripts derived from the AV3 (42), TOM (43) and Dynamo packages (44). Initial angles were assigned according to the geometry of a sphere. A starting reference generated from a preliminary dataset containing density for the CA-SP1-NC region and filtered to 32 Å was used for both untreated ΔMACANCSP2 and ΔMACANCSP2+BVM datasets. A starting reference for the immature HIV-1 D25A dataset was generated from a single tomogram. 6-fold symmetry inherent in the structures was applied throughout processing.

For the VLP datasets two rounds of alignment and averaging were performed. Subsequently, sub-volumes that had converged onto the same position or that contained no protein density corresponding to the CA-SP1 layer were removed using a “sub-volume to sub-volume distance” and cross-correlation threshold, respectively. The remaining sub-volumes were aligned for one (untreated ΔMACANCSP2) or two more (ΔMACANCSP2+BVM) iterations.

For the HIV-1 D25A virus dataset four iterations of alignment and averaging were performed to determine the initial orientations and positions of the sub-volumes on the lattice. After the first three rounds, sub-volumes that converged onto the same position of the Gag lattice were removed using a “sub-volume to sub-volume distance”. After another iteration sub-volumes that contained no Gag protein density were removed according to their lower cross-correlation values.

Sub-volumes of a size of (389)3 Å were then extracted from 4x-binned tomograms at positions determined in the 8x-binned alignments and averages were generated by aligning the sub-volumes according to angles determined in the 8x-binned alignments. The datasets were aligned for three iterations, with a progressive reduction in angular search. During alignment, a low-pass filter was applied at 26 Å resolution for the untreated ΔMACANCSP2 dataset and 32 Å for the ΔMACANCSP2+BVM and HIV-1 (D25A) virus datasets. At the end of the 4x-binned alignments a “sub-volume to sub-volume distance” threshold was again applied. The remaining sub-volumes with a box size of (346)3 Å were extracted at their aligned positions from 2x-binned tomograms. At this stage, the dataset was split into even/odd half datasets and from this stage on, even/odd datasets were treated absolutely independently. Sub-volumes with mean grey

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values further than ±1x standard deviation from the dataset mean (e.g. containing parts of gold particles) were removed.

For the even/odd datasets independent references were generated by averaging their respective sub-volumes and filtered to 17 Å. After two more rounds of alignment with sequentially reduced angular searches, sub-volumes of size (259)3 Å were finally extracted from unbinned tomograms. Independent references were generated from sub-volumes of each respective dataset. Low-pass filters were set to 8.1 Å and sub-volumes were aligned two more rounds using only 6x1 degree angular sampling cones (out of plane and in plane). Final averages were generated from 265,506/263,910; 386,040/386,598; and 301,302/301,920 asymmetric units in the even/odd halfsets of the untreated ΔMACANCSP2, ΔMACANCSP2+BVM and immature HIV-1 (D25A) virus data, respectively.

The final averages were multiplied with a Gaussian-filtered cylindrical mask and resolution was determined by mask-corrected Fourier-shell correlation (45). The half maps were averaged and reweighted by division by their summed CTFs. Finally the untreated ΔMACANCSP2, ΔMACANCSP2+BVM and HIV-1 (D25A) virus maps were sharpened with an empirically determined B-factor of -490, -350 or -380 A2, respectively and filtered to their measured resolutions (46). The structures displayed α-helices showing helical pitches and clear densities for aromatic, basic and even smaller hydrophobic side chains consistent with the measured resolutions.

To calculate the difference maps shown in Fig. S2G-I, the amplitude spectra of the respective maps were scaled, the grey-values of the maps were normalized between 20 and 6 Å, the maps filtered to 6 Å resolution, and a high-pass filter applied at 130 Å to remove any low-frequency gradients. They were then subtracted from each other to generate the difference maps.

EM-densities were visualized in Chimera (47), Coot (48) and Pymol (49).

Improvement in resolution

The following differences in data acquisition and image processing contributed to the increase in resolution from ~8 Å in our previously published structure of immature HIV-1 (6) to ~4 Å for the structures presented here. We have collected data using a Gatan Quantum 967 LS energy filter with Gatan K2xp direct detector, compared to a Gatan GIF2002 energy filter with CCD camera. The direct electron detector gives significantly improved detective quantum efficiency, and therefore better image quality. The data was collected at 300kV instead of 200kV. The larger camera field of view allowed more efficient collection of a larger dataset. Movie-mode processing was applied to compensate for beam-induced motion (14). Improved image quality allowed per-tilt defocus-determination. Use of an optimized dose-symmetric tilt scheme for data collection improved data quality by redistributing dose-dependent sample damage to the higher-tilts, thereby improving high-resolution information transfer and minimizing the impact of does-dependent sample distortions (12). This allowed application of a higher

6

electron dose. The introduction of exposure filtering (15) further improved the signal to noise ratio in reconstructed tomograms.

Atomic model building and refinement

All refinement was done in the 3.9 Å resolution ΔMACANCSP2+BVM map. In order to obtain a starting model for coordinate refinement, the NMR model of the CA-NTD (PDB 1L6N, chain 1, residues 148-279) (16) and the crystal structure of CA-CTD (PDB 3DS2, one monomer, residues 280-353) (19) were rigid body docked into the EM-density of one CA-monomer using the “Fit in map” option in Chimera (47). A starting model for the CA-SP1 region was obtained by extracting residues 354-371 from a solution structure of a CA-SP1 peptide (PDB 1U57) (50), in which residues 351-357 are disordered, and 358-380 form a helix. Chain breaks were joined in Coot (48) and residue Ala301 in PDB 3DS2 was restored to Tyr301 as in the wild-type sequence.

To account for all possible monomer-monomer interactions in CA, a map segment corresponding to six CA monomers was extracted from the EM density using a mask extending 3 Å outwards from the center of the rigid-body-fitted model coordinates. We then performed automated real-space coordinate refinement against the EM density using a real-space refinement workflow (51, 52) based on CCP4 (53) and cctbx/ PHENIX (54, 55) modules, which was iterated with manual model building in Coot (48) until convergence.

Briefly, the workflow is as follows. Map segment and model were centered in a cubic box of P1 symmetry with a cell edge of 324 Å to allow uniform grid sampling of model and experimental maps at the experimental pixel size. The Cyclophilin-A binding loop region (residues 216-232) displayed weak density suggestive of multiple conformations. A single conformation with maximum real-space correlation was obtained after density-guided model deformation (morphing). This conformation was restrained with a weak harmonic potential in subsequent refinement cycles to scale the global refinement weights to the poor local resolution in this area. Individual isotropic atomic displacement parameters (ADPs) were set to 60 Å2 at the beginning of refinement. Real-space coordinate and ADP refinement was then performed using gradient-driven minimization of a combined map and restraint target as implemented in PHENIX (54). Non-crystallographic symmetry restraints and secondary structure restraints were applied throughout. Secondary structure restraints were interrogated and updated during each refinement iteration based on CaBLAM (56) analysis. Weights on density and geometry restraints were optimized during each refinement cycle. Each round of model optimization was evaluated by computing the real-space cross-correlation (RSCC) between experimental map and a model map simulated by calculating B-factor-weighted structure factors from the model coordinates. Electron atomic form factors (57) were used in computation of structure factors. At the end of each iteration, the central monomer of this assembly was used for generation of the symmetrized model of the biological assembly as the starting point for the next iteration. The disulfide bond between Cys330 and Cys350 in the CA-CTD was not modeled, but the EM-density suggests it may be partially occupied.

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For cross-validation of the final model, model bias was removed by random coordinate displacement up to a maximum of 0.4 Å, followed by 5 cycles of real-space refinement against one of the half map reconstructions (work map) using the same refinement parameters as determined above. We tested for model overfitting by computing the FSC between the model and the work map (FSCwork) or model and the second half map not used for coordinate refinement (FSCtest) (Fig. S3A). Significant deviations between both curves would be a sign of overfitting. The quality of the final model was validated using MOLPROBITY (58) and was found to range in the top percentile for the corresponding resolution range.

Comparison of the EM densities of the three samples by FSC (Fig. S2B), by difference mapping (Fig S2D-I), and by visual inspection (Fig. S3C) suggested that there were no substantial differences in protein structure. To identify any small local differences in protein structure we refined the ΔMACANCSP2+BVM model into the EM densities of the untreated ΔMACANCSP2 and immature HIV-1 (D25A) samples as above to generate structural models for those samples. We then calculated the root mean square deviations (RMSDs) in atom position between the ΔMACANCSP2+BVM model and these new models. C-alpha RMSDs were 0.49 Å and 0.48 Å, and all-atom RMSDs were 1.14 Å and 1.21 Å respectively. Residue-by-residue all-atom RMSDs were below 1 Å for all residues except six in untreated ΔMACANCSP2, and three in immature HIV-1 (D25A), which are located in intrinsically flexible regions or can be attributed to resolution variation between the maps. We conclude that at the determined resolution there are no substantial differences in protein structure between the three samples.

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Fig. S1. Schematic representation of the late stage of the HIV-1 lifecycle and the structure of Gag. (A) The late stage of the virus lifecycle consists of assembly, budding and maturation. Gag oligomerization starts in the cytoplasm and is driven by protein contacts mediated by the CA domains and genome-NC interactions. Gag oligomers then shuttle to the plasma membrane and form hexameric lattices. Upon budding the virus forms roughly spherical particles of varying dimensions with the Gag lattice underlining the viral membrane in the shape of a truncated sphere with irregular defects. During maturation, proteolytic cleavage by the viral protease causes the separation of the individual Gag domains and rearrangement of the virus structure into the mature

Extracellular

Cytoplasm

Budding

Maturation

Assembly

Protease and Maturation inhibitors

CA

NC p6

SP2

NC

CASP1

MA CA NCSP1 SP2

p655kDa Gag

MA CASP1

NCSP2

MA CASP1

NC p6SP2

Ma

tura

tio

n in

hib

ito

rs

A B

HIV-1 CA-NTD CA-CTD NC p6MA

SP1 SP2

1 132 363 377 432 448 500у0$&$1&63�

133 192PIVQNLQGQM VHQAISPRTL NAWVKVVEEK AFSPEVIPMF SALSEGATPQ DLNTMLNTVG

PATIM

373 377

193 252GHQAAMQMLK ETINEEAAEW DRLHPVHAGP IAPGQMREPR GSDIAGTTST LQEQIGWMTH

312253NPPIPVGEIY KRWIILGLNK IVRMYSPTSI LDIRQGPKEP FRDYVDRFYK TLRAEQASQE

313 372VKNWMTETLL VQNANPDCKT ILKALGPGAT LEEMMTACQG VGGPGHKARV LAEAMSQVTN

H1 H2 H3

H4 H6

H7 H8

H9 H11

H5

H10

310 H9

CypA-BL

CA-SP1 Helix

C

D

9

infectious form. Maturation can be blocked by PR inhibitors and MIs. (B) The ordered cascade of cleavage at the five cleavage sites in Gag. The final cleavage, between CA and SP1, is blocked by the maturation inhibitors. (C) Schematic representation of the HIV-1 Gag domain composition. The extent of the ΔMACANCSP2 construct is shown with black rectangles. (D) Sequence of HIV-1 CA-SP1. The positions of α-helices are shown as cyan and yellow bars. The Cyclophilin A binding loop is indicated with a cyan ellipsoid. Regions for which published structural data is lacking are dashed. Triangles denote protease cleavage sites. The final proteolytic cleavage during maturation occurs between L363 and A364.

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Fig. S2 Comparisons of cryo-electron tomography and subtomogram averaging reconstructions of the immature HIV-1 CA-SP1 lattice. (A-B) Fourier Shell correlations (FSC) between independent half datasets for untreated ΔMACANCSP2

11

(red), ΔMACANCSP2+BVM (blue) and immature HIV-1 (D25A) (green). Measured resolutions are marked in the figure. (B) FSCs between ΔMACANCSP2+BVM and untreated ΔMACANCSP2 structures (green), between ΔMACANCSP2+BVM and immature HIV-1 (D25A) solved in this study (light blue) and between ΔMACANCSP2+BVM and the previously solved 8.8 Å structure of immature HIV-1 CA (EMD-2706) (purple). (C) Isosurface comparison of the CA-SP1 reconstructions from the VLPs and the PR defective (D25A) virus obtained in this study to the previously solved CA-SP1 structure from within PR-inhibited immature HIV-1 viruses (emd-2706). When filtered to the comparable resolution of 8.8 Å, all CA-SP1 structures are the same. (D-F) Left and right panels show radial orthoslices through the final unsharpened CA-SP1 averages of untreated ΔMACANCSP2, ΔMACANCSP2 + BVM and protease defective HIV-1 (D25A) filtered between 130 Å and 6 Å. Density is white. Scale bars are 25 Å. (G-I) Left hand panels show difference maps calculated by subtracting the structures in the left panels of D-F from those in the right panels of D-F (13). Right hand panels shows the difference maps in three-dimensions in red. The isosurface of the untreated ΔMACANCSP2 structure is shown in grey for orientation purposes. Overall, no clear differences in the CA-SP1 structure are observed (see also (13)). Differences are seen at the position of the density coordinated by the lysine rings (arrowhead) (K290 and K359, Fig 2F) and in the center of the six-helix bundle (arrow). The density coordinated by the lysine rings is present in all samples but shows differences in the three maps (arrowheads). This density appears more compact upon addition of BVM to the VLPs, giving rise to the adjacent positive and negative densities in G. This density appears much weaker in the PR defective HIV-1 sample than in the VLPs, giving rise to a positive signal at this position in the difference maps in H and I. Additional density is seen in the center of the CA-SP1 6-helix bundle in the +BVM sample (arrow), that gives rise to a positive signal at this position in the difference maps in G and I. The density located at the center of the lysine rings is presumably contributed by negatively charged small molecules or ions coordinated by these rings. This density cannot be explained as an experimental artifact because it is seen at all equivalent six-helix bundles within each solved structure, not just at the central one where six-fold symmetry is applied, and because it is seen at this position in our previous 8.8 Å structure (6), and in the 8 Å resolution structure of immature-like tubular arrays solved by helical reconstruction (59). We hypothesize that the molecules coordinated at this position differ between the immature virus and the in vitro assembled VLPs leading to the weaker density in the virus. We attribute the additional density at the center of the six-helix bundle in the +BVM sample to BVM in its binding site. This density is seen at all equivalent six-helix bundles within the solved structure. An asymmetric molecule located directly on the 6-fold access will be smeared-out by the application of 6-fold symmetry, and the orientation of the molecule therefore cannot be defined.

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Fig. S3 Refinement of the immature HIV-1 CA-SP1 model. (A) FSCs between the refined model and the half map used for refinement (FSCwork, blue), between the refined model and the other half map (FSCtest, green) and between the refined model and the full map (FSCref, red). The similarity of the FSCwork and FSCtest curves indicate that the model is not over-refined. (B) Distribution of B-factors in the refined model. Flexible regions, in particular the Cyclophilin A binding loop (residues 216-232), have higher B-factors (red), correlating with lower local map resolution due to increased structural flexibility. (C) Representative EM-densities for immature HIV-1, untreated ΔMACANCSP2, and ΔMACANCSP2 + BVM, superimposed with the model refined against the 3.9 Å ΔMACANCSP2 + BVM map. The model is consistent with all three structures, illustrating the similarity in the protein structure in the three samples. The densities for ΔMACANCSP2 + BVM are shown at different isosurface thresholds to illustrate the high quality of the density. The α-helices show helical pitches and clear densities for aromatic, basic and even smaller hydrophobic side chains.

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Fig. S4 Contacts within the CA-NTD layer, and at the CA-CTD dimer interface. (A) The CA-NTD layer viewed from outside the particle. One CA-NTD monomer is highlighted and the refined side chain positions are shown. The general arrangement of the CA-NTD is consistent with previous models (6). α-helices are annotated and symmetry axes are marked. Colored rectangles indicate interfaces shown in the insets. The inter-hexamer interactions are formed by extensive two-fold and three-fold interfaces and appear to be stabilized by a salt bridge between residues E207/E208 in helix 4 (mutation of which interferes with assembly (22)) and R150 in helix 1 of the three-fold related capsid monomer. Stabilizing contacts around the hexameric ring involve R214 (helix 4), which projects towards N185 and T186 (helix 3) (B) The CA-CTD layer viewed as in (A). The highly conserved hydrophobic residues in helix 9 (W316 and M317) form the dimeric CA-CTD interface. These are the same residues that form the dimeric CA-CTD interface in the mature virus.

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Fig. S5 In vitro cleavage of ΔMACANCSP2 VLPs. (A) Western blot analysis of untreated ΔMACANCSP2 and ΔMACANCSP2 + BVM VLP cleavage by recombinant HIV-1 PR (13). (B) CA and CA-SP1 bands were quantified using ImageJ and accumulation of CA-SP1 cleavage intermediates were calculated as ratio of band intensities: CA-SP1 / (CA-SP1+CA). ** denotes p<0.01, error bars denote standard deviation, N=3. BVM leads to a delay in cleavage between CA and CA-SP1.

A

B

Untreated +BVM

CA-SP1CA

50

60

70

80

untreated +BVM

**

% C

A-SP

1 ac

cum

ulat

ion

15

Table S1. Summary of Data acquisition and Image processing statistics

Sample HIV-1 ΔMACANCSP2 VLPs

HIV-1 ΔMACANCSP2 VLPs

+ 100 µg/ml Bevirimat Immature HIV-1 (D25A)

virus

Acquisition settings Microscope FEI Titan Krios FEI Titan Krios FEI Titan Krios

Voltage (keV) 300 300 300

Detector Gatan Quantum K2 Gatan Quantum K2 Gatan Quantum K2

Energy-filter Yes Yes Yes

Slit width (eV) 20 20 20

Super-resolution mode Yes Yes Yes

Å/pixel 1.35 1.35 1.35

Defocus range (microns) -1.5 to -4.5

-1.5 to -5.0 -1.5 to 5.0

Defocus step (microns) 0.25 0.25 0.25

Acquisition scheme -60/60°, 3°, Serial EM -60/60°, 3°, Serial EM -60/60°, 3°, Serial EM

Total Dose (electrons/Å2) ~90 - 270 ~120 - 145 ~120-221

Dose rate (electrons/Å2/sec)

~3 - 8 ~3 - 3.8 ~1.5 – 5.5

Frame number 6 – 10 8 – 10 10 – 12

Tomogram number 93 43 74

Processing settings VLPs/Viruses 285 383 484

Asymmetric units Set A 265,506 386,040 301,302

Asymmetric units Set B 263,910 386,598 301,920

Final resolution (0.143 FSC) in Å

4.5 3.9 4.2

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Table S2. Summary of model refinement statistics

Refinement HIV-1 ΔMACANCSP2 Residues 143-373

Number of residues 223

All-atom clash score

2.34

Favored rotamers 97.61%

Ramachandran outliers 0

Ramachandran favored 90.24%

C-beta deviations 0

Rmsd (angles, degree) 0.72

Rmsd (bonds ,Å) 0.003

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Movie S1 A 3D-visualization of the HIV-1 CA-SP1 structure at 3.9 Å resolution.

Movie S2 A guided tour through the structure highlighting structural features presented in this study.

References and Notes 1. W. I. Sundquist, H. G. Kräusslich, HIV-1 assembly, budding, and maturation. Cold

Spring Harb. Perspect. Med. 2, a006924 (2012). Medline doi:10.1101/cshperspect.a015420

2. B. Müller, M. Anders, H. Akiyama, S. Welsch, B. Glass, K. Nikovics, F. Clavel, H.-M. Tervo, O. T. Keppler, H.-G. Kräusslich, HIV-1 Gag processing intermediates trans-dominantly interfere with HIV-1 infectivity. J. Biol. Chem. 284, 29692–29703 (2009). Medline doi:10.1074/jbc.M109.027144

3. M. A. Checkley, B. G. Luttge, F. Soheilian, K. Nagashima, E. O. Freed, The capsid-spacer peptide 1 Gag processing intermediate is a dominant-negative inhibitor of HIV-1 maturation. Virology 400, 137–144 (2010). Medline doi:10.1016/j.virol.2010.01.028

4. C. S. Adamson, K. Salzwedel, E. O. Freed, Virus maturation as a new HIV-1 therapeutic target. Expert Opin. Ther. Targets 13, 895–908 (2009). Medline doi:10.1517/14728220903039714

5. C. S. Adamson, M. Sakalian, K. Salzwedel, E. O. Freed, Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1maturation inhibitor bevirimat. Retrovirology 7, 36 (2010). doi:10.1186/1742-4690-7-36

6. F. K. Schur, W. J. Hagen, M. Rumlová, T. Ruml, B. Müller, H. G. Kräusslich, J. A. Briggs, Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution. Nature 517, 505–508 (2015). Medline doi:10.1038/nature13838

7. J. A. Briggs, Structural biology in situ—the potential of subtomogram averaging. Curr. Opin. Struct. Biol. 23, 261–267 (2013). Medline doi:10.1016/j.sbi.2013.02.003

8. M. A. Accola, S. Höglund, H. G. Göttlinger, A putative α-helical structure which overlaps the capsid-p2 boundary in the human immunodeficiency virus type 1 Gag precursor is crucial for viral particle assembly. J. Virol. 72, 2072–2078 (1998). Medline

9. S. A. Datta, L. G. Temeselew, R. M. Crist, F. Soheilian, A. Kamata, J. Mirro, D. Harvin, K. Nagashima, R. E. Cachau, A. Rein, On the role of the SP1 domain in HIV-1 particle assembly: A molecular switch? J. Virol. 85, 4111–4121 (2011). Medline doi:10.1128/JVI.00006-11

10. A. de Marco, B. Müller, B. Glass, J. D. Riches, H.-G. Kräusslich, J. A. G. Briggs, Structural analysis of HIV-1 maturation using cryo-electron tomography. PLOS Pathog. 6, e1001215 (2010). doi:10.1371/journal.ppat.1001215

11. I. Gross, H. Hohenberg, T. Wilk, K. Wiegers, M. Grättinger, B. Müller, S. Fuller, H. G. Kräusslich, A conformational switch controlling HIV-1 morphogenesis. EMBO J. 19, 103–113 (2000). Medline doi:10.1093/emboj/19.1.103

12. W. J. Hagen, W. Wan, J. A. Briggs, Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 10.1016/j.jsb.2016.06.007 (2016). Medline doi:10.1016/j.jsb.2016.06.007

13. Materials and methods are available as supplementary materials on Science Online.

14. X. Li, P. Mooney, S. Zheng, C. R. Booth, M. B. Braunfeld, S. Gubbens, D. A. Agard, Y. Cheng, Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013). Medline doi:10.1038/nmeth.2472

15. T. Grant, N. Grigorieff, Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015). Medline doi:10.7554/eLife.06980

16. C. Tang, Y. Ndassa, M. F. Summers, Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein. Nat. Struct. Biol. 9, 537–543 (2002). Medline

17. A. T. Gres, K. A. Kirby, V. N. KewalRamani, J. J. Tanner, O. Pornillos, S. G. Sarafianos, X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability. Science 349, 99–103 (2015). Medline doi:10.1126/science.aaa5936

18. J. A. Briggs, J. D. Riches, B. Glass, V. Bartonova, G. Zanetti, H. G. Kräusslich, Structure and assembly of immature HIV. Proc. Natl. Acad. Sci. U.S.A. 106, 11090–11095 (2009). Medline doi:10.1073/pnas.0903535106

19. V. Bartonova, S. Igonet, J. Sticht, B. Glass, A. Habermann, M. C. Vaney, P. Sehr, J. Lewis, F. A. Rey, H. G. Kraüsslich, Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein. J. Biol. Chem. 283, 32024–32033 (2008). Medline doi:10.1074/jbc.M804230200

20. T. R. Gamble, S. Yoo, F. F. Vajdos, U. K. von Schwedler, D. K. Worthylake, H. Wang, J. P. McCutcheon, W. I. Sundquist, C. P. Hill, Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. Science 278, 849–853 (1997). Medline doi:10.1126/science.278.5339.849

21. O. Pornillos, B. K. Ganser-Pornillos, B. N. Kelly, Y. Hua, F. G. Whitby, C. D. Stout, W. I. Sundquist, C. P. Hill, M. Yeager, X-ray structures of the hexameric building block of the HIV capsid. Cell 137, 1282–1292 (2009). Medline doi:10.1016/j.cell.2009.04.063

22. U. K. von Schwedler, K. M. Stray, J. E. Garrus, W. I. Sundquist, Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J. Virol. 77, 5439–5450 (2003). Medline doi:10.1128/JVI.77.9.5439-5450.2003

23. D. Melamed, M. Mark-Danieli, M. Kenan-Eichler, O. Kraus, A. Castiel, N. Laham, T. Pupko, F. Glaser, N. Ben-Tal, E. Bacharach, The conserved carboxy terminus of the capsid domain of human immunodeficiency virus type 1 Gag protein is important for virion assembly and release. J. Virol. 78, 9675–9688 (2004). Medline doi:10.1128/JVI.78.18.9675-9688.2004

24. M. Prabu-Jeyabalan, E. Nalivaika, C. A. Schiffer, Substrate shape determines specificity of recognition for HIV-1 protease: Analysis of crystal structures of six substrate complexes. Structure 10, 369–381 (2002). Medline doi:10.1016/S0969-2126(02)00720-7

25. K. Wiegers, G. Rutter, H. Kottler, U. Tessmer, H. Hohenberg, H.-G. Kräusslich, Sequential steps in human immunodeficiency virus particle maturation

revealed by alterations of individual Gag polyprotein cleavage sites. J. Virol. 72, 2846–2854 (1998). Medline

26. M. Sakalian, C. P. McMurtrey, F. J. Deeg, C. W. Maloy, F. Li, C. T. Wild, K. Salzwedel, 3-O-(3′,3′-dimethysuccinyl) betulinic acid inhibits maturation of the human immunodeficiency virus type 1 Gag precursor assembled in vitro. J. Virol. 80, 5716–5722 (2006). doi:10.1128/JVI.02743-05

27. P. W. Keller, R. K. Huang, M. R. England, K. Waki, N. Cheng, J. B. Heymann, R. C. Craven, E. O. Freed, A. C. Steven, A two-pronged structural analysis of retroviral maturation indicates that core formation proceeds by a disassembly-reassembly pathway rather than a displacive transition. J. Virol. 87, 13655–13664 (2013). Medline doi:10.1128/JVI.01408-13

28. P. W. Keller, C. S. Adamson, J. B. Heymann, E. O. Freed, A. C. Steven, HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice. J. Virol. 85, 1420–1428 (2011). Medline doi:10.1128/JVI.01926-10

29. C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, E. O. Freed, In vitro resistance to the human immunodeficiency virus type 1 maturation inhibitor PA-457 (bevirimat). J. Virol. 80, 10957–10971 (2006). Medline doi:10.1128/JVI.01369-06

30. N. A. Margot, C. S. Gibbs, M. D. Miller, Phenotypic susceptibility to bevirimat in isolates from HIV-1-infected patients without prior exposure to bevirimat. Antimicrob. Agents Chemother. 54, 2345–2353 (2010). Medline doi:10.1128/AAC.01784-09

31. K. Waki, S. R. Durell, F. Soheilian, K. Nagashima, S. L. Butler, E. O. Freed, Structural and functional insights into the HIV-1 maturation inhibitor binding pocket. PLOS Pathog. 8, e1002997 (2012). Medline doi:10.1371/journal.ppat.1002997

32. W. S. Blair, J. Cao, J. Fok-Seang, P. Griffin, J. Isaacson, R. L. Jackson, E. Murray, A. K. Patick, Q. Peng, M. Perros, C. Pickford, H. Wu, S. L. Butler, New small-molecule inhibitor class targeting human immunodeficiency virus type 1 virion maturation. Antimicrob. Agents Chemother. 53, 5080–5087 (2009). Medline doi:10.1128/AAC.00759-09

33. J. M. Wagner, K. K. Zadrozny, J. Chrustowicz, M. D. Purdy, M. Yeager, B. K. Ganser-Pornillos, O. Pornillos, Crystal structure of an HIV assembly and maturation switch. eLife 5, e17063 (2016). Medline doi:10.7554/eLife.17063

34. M. Dettenhofer, X.-F. Yu, Highly purified human immunodeficiency virus type 1 reveals a virtual absence of Vif in virions. J. Virol. 73, 1460–1467 (1999). Medline

35. D. N. Mastronarde, Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005). Medline doi:10.1016/j.jsb.2005.07.007

36. M. Vulović, E. Franken, R. B. G. Ravelli, L. J. van Vliet, B. Rieger, Precise and unbiased estimation of astigmatism and defocus in transmission electron microscopy. Ultramicroscopy 116, 115–134 (2012). doi:10.1016/j.ultramic.2012.03.004

37. A. Rohou, N. Grigorieff, CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). Medline doi:10.1016/j.jsb.2015.08.008

38. F. K. Schur, W. J. Hagen, A. de Marco, J. A. Briggs, Determination of protein structure at 8.5Å resolution using cryo-electron tomography and sub-tomogram averaging. J. Struct. Biol. 184, 394–400 (2013). Medline doi:10.1016/j.jsb.2013.10.015

39. J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996). Medline doi:10.1006/jsbi.1996.0013

40. Q. Xiong, M. K. Morphew, C. L. Schwartz, A. H. Hoenger, D. N. Mastronarde, CTF determination and correction for low dose tomographic tilt series. J. Struct. Biol. 168, 378–387 (2009). Medline doi:10.1016/j.jsb.2009.08.016

41. S. Pruggnaller, M. Mayr, A. S. Frangakis, A visualization and segmentation toolbox for electron microscopy. J. Struct. Biol. 164, 161–165 (2008). Medline doi:10.1016/j.jsb.2008.05.003

42. F. Förster, O. Medalia, N. Zauberman, W. Baumeister, D. Fass, Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography. Proc. Natl. Acad. Sci. U.S.A. 102, 4729–4734 (2005). Medline doi:10.1073/pnas.0409178102

43. S. Nickell, F. Förster, A. Linaroudis, W. D. Net, F. Beck, R. Hegerl, W. Baumeister, J. M. Plitzko, TOM software toolbox: Acquisition and analysis for electron tomography. J. Struct. Biol. 149, 227–234 (2005). Medline doi:10.1016/j.jsb.2004.10.006

44. D. Castaño-Díez, M. Kudryashev, M. Arheit, H. Stahlberg, Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J. Struct. Biol. 178, 139–151 (2012). Medline doi:10.1016/j.jsb.2011.12.017

45. S. Chen, G. McMullan, A. R. Faruqi, G. N. Murshudov, J. M. Short, S. H. W. Scheres, R. Henderson, High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013). Medline doi:10.1016/j.ultramic.2013.06.004

46. P. B. Rosenthal, R. Henderson, Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003). Medline doi:10.1016/j.jmb.2003.07.013

47. E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, T. E. Ferrin, UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). Medline doi:10.1002/jcc.20084

48. P. Emsley, K. Cowtan, Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004). Medline doi:10.1107/S0907444904019158

49. The PyMOL Molecular Graphics System (Schrodinger, 2010).

50. N. Morellet, S. Druillennec, C. Lenoir, S. Bouaziz, B. P. Roques, Helical structure determined by NMR of the HIV-1 (345–392)Gag sequence, surrounding p2: Implications for particle assembly and RNA packaging. Protein Sci. 14, 375–386 (2005). Medline doi:10.1110/ps.041087605

51. N. A. Hoffmann, A. J. Jakobi, M. Moreno-Morcillo, S. Glatt, J. Kosinski, W. J. H. Hagen, C. Sachse, C. W. Müller, Molecular structures of unbound and transcribing RNA polymerase III. Nature 528, 231–236 (2015). Medline doi:10.1038/nature16143

52. S. A. Fromm, T. A. Bharat, A. J. Jakobi, W. J. Hagen, C. Sachse, Seeing tobacco mosaic virus through direct electron detectors. J. Struct. Biol. 189, 87–97 (2015). Medline doi:10.1016/j.jsb.2014.12.002

53. M. D. Winn, C. C. Ballard, K. D. Cowtan, E. J. Dodson, P. Emsley, P. R. Evans, R. M. Keegan, E. B. Krissinel, A. G. W. Leslie, A. McCoy, S. J. McNicholas, G. N. Murshudov, N. S. Pannu, E. A. Potterton, H. R. Powell, R. J. Read, A. Vagin, K. S. Wilson, Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011). Medline doi:10.1107/S0907444910045749

54. P. D. Adams, P. V. Afonine, G. Bunkóczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart, PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). Medline doi:10.1107/S0907444909052925

55. R. W. Grosse-Kunstleve, N. K. Sauter, N. W. Moriarty, P. D. Adams, The Computational Crystallography Toolbox: Crystallographic algorithms in a reusable software framework. J. Appl. Cryst. 35, 126–136 (2002). doi:10.1107/S0021889801017824

56. C. J. Williams, B. J. Hintze, D. C. Richardson, J. S. Richardson, CaBLAM: Identification and scoring of disguised secondary structure at low resolution. Comput. Crystallogr. Newsl. 4, 33 (2013).

57. C. Colliex, J. M. Cowley, S. L. Dudarev, M. Fink, J. Gjønnes, R. Hilderbrandt, A. Howie, D. F. Lynch, L. M. Peng, G. Ren, A. W. Ross, V. H. Smith, J. C. H. Spence, J. W. Steeds, J. Wang, M. J. Whelan, B. B. Zvyagin, “Electron diffraction,” in International Tables for Crystallography, Volume C: Mathematical, Physical, and Chemical Tables, E. Prince, Ed. (International Union of Crystallography, 2006), chap. 4.3, pp. 259–429.

58. V. B. Chen, W. B. Arendall 3rd, J. J. Headd, D. A. Keedy, R. M. Immormino, G. J. Kapral, L. W. Murray, J. S. Richardson, D. C. Richardson, MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010). Medline doi:10.1107/S0907444909042073

59. T. A. Bharat, L. R. Castillo Menendez, W. J. Hagen, V. Lux, S. Igonet, M. Schorb, F. K. Schur, H. G. Kräusslich, J. A. Briggs, Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly. Proc. Natl. Acad. Sci. U.S.A. 111, 8233–8238 (2014). Medline doi:10.1073/pnas.1401455111