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Supporting InformationBhattacharya et al.
10.1073/pnas.1419945112SI Materials and MethodsStructure
Determination of the PF74-Bound CA Hexamer.Diffractiondata were
collected from several soaked crystals at beamline22-BM at the
Advanced Photon Source, Argonne NationalLaboratory. Data were
indexed, integrated, and scaled withHKL2000. The unbound
isomorphous structure (PDB ID: 3H47)was used as a starting model
for rigid body refinement. A data setthat provided clear positive
difference density that unambiguouslydefined the conformation of
the bound ligand was used forsubsequent rounds of iterative model
building [performed withCOOT (1)] and refinement [performed with
PHENIX, version1.9-1692 (2)]. Data and structure statistics are
shown in Table S1.
Structure Determination of the CPSF6-Bound CA Hexamer.
Crystalsfrom Molecular Dimensions Morpheus Suite condition
#2-31[0.1 M sodium formate, ammonium acetate, tri-sodium
citrate,sodium/potassium tartrate, sodium oxamate; 0.1 M
sodiumHepes, MOPS (pH 7.5); 30% glycerol, PEG4000] were
harvestedand flash-frozen in liquid nitrogen before data collection
atbeamline 24-ID-C at the Advanced Photon Source, Argonne Na-tional
Laboratory. Diffraction data were processed using X-rayDetector
Software (available from the Max Planck Institute forMedical
Research, Heidelberg, Germany). Phases were generatedusing the
isomorphous CA hexamer structure (PDB ID: 3H4E).Coordinates were
refined using PHENIX (version 1.9-1692) withtorsion angle simulated
annealing (2). Manual model buildingand rebuilding was performed
with COOT (1). Noncrystallographicsymmetry restraints were applied
throughout the refinement pro-cess. Data collection and refinement
statistics are shown in Table S1.
“Fate of the Capsid” Assay. The assay was performed according
toref. 3, as first described by Stremlau et al. (4). Stably
transducedHeLa (1.5 × 106) cells expressing the indicated proteins
wereseeded in 80-cm2 flasks. The following day, cells were
incubatedwith 5–10 mL (2.5–5.0 × 105 reverse transcriptase units)
of HIV-1-GFP at 4 °C for 30 min to allow viral attachment to the
cells.The cells were then shifted to 37 °C until they were
harvested12 h after infection. Cells were washed three times using
ice-coldPBS and detached by treatment with 1.0 mL of pronase (7.0
mg/mLin DMEM) for 5 min at 25 °C. The cells were then washedthree
times with PBS. The cells were resuspended in 2.5 mLhypotonic lysis
buffer [10 mM Tris·HCl (pH 8.0), 10 mM KCl,1 mM EDTA, and one
complete protease inhibitor tablet] andincubated on ice for 15 min.
The cells were lysed using 15 strokesin a 7.0-mL Dounce homogenizer
with pestle B. Cellular debriswas cleared by centrifugation for 3
min at 3,000 rpm. To allowassessment of the INPUT for HIV-1 p24,
100 μL of the clearedlysate were collected, made 1× in SDS sample
buffer, and ana-lyzed by Western immunoblot analysis. Then 2.0 mL
of thecleared lysate were layered onto a 50% sucrose
(weight:volume)cushion in 1× PBS and centrifuged at 125,000 × g for
2 h at 4 °Cin a Beckman SW41 rotor. After centrifugation, 100 μL of
thetop-most portion of the supernatant were collected and made
1× in SDS sample buffer; this sample is referred to as
theSOLUBLE fraction. The PELLET was resuspended in 50 μL 1×SDS
sample buffer and is referred to as the particulate. INPUT,SOLUBLE,
and PELLET samples were then subjected toSDS/PAGE and Western
immunoblot analysis (Fig. 2B). TheHIV-1 p24 proteins were detected
using a mouse anti-p24 anti-body (Immuno Diagnostics).
Isothermal Titration Calorimetry. Protein samples were first
ex-tensively dialyzed against PBS buffer (137 mM NaCl, 2.7 mMKCl,
10 mM Na2HPO4, and 2 mM KH2PO4) over 3 d and thendiluted to working
concentrations between 300 μM and 1 mM toyield sigmoidal binding
curves depending on the observed af-finity range. A stock solution
of PF74 (50 mM in DMSO) wasdiluted to 30 μM in the same buffer.
DMSO was also added tothe diluted proteins to match the PF74 stock
(0.07%) to avoidartifacts from heats of dilution of DMSO, and both
the PF74 andprotein solutions were degassed and centrifuged before
titration.Protein solutions were injected (22 injections of 1.8 μL
at 3-minintervals) into a cell chamber containing 207 μL of PF74
solution(30 μM). Baseline controls were acquired by injecting CA
pro-tein solutions into a cell chamber filled with
DMSO-matchedbuffer and then subtracted from the respective
experimental ti-trations before data fitting. Isotherms were fit to
a single-sitemodel with MicroCal’s ORIGIN software, assuming a
singlePF74 binding site in each CA monomer. CA protein
concen-trations in Figs. S2 and S3 and Table S2 are expressed in
termsof monomeric protein equivalents. Titrations were performed
intriplicate, and the averaged fitting results are reported in
Table S2.
Analytical Ultracentrifugation. Fluorescent CPSF6313–327
(PVLF-PGQPFGQPPLG) and NUP1531407–1429 (TNNSPSGVFTFG-ANSSTPAASAQ)
peptides were synthesized with N-terminalFITC β-Ala fluorescent
tags at the Tufts University Core Facil-ity. The CPSF6 peptide also
contained a C-terminal hydrophilictail (SKGSKS). Experiments were
performed in 50 mM Tris (pH8) and 50 mM NaCl. The fluorescence
optics system consisted ofa 488-nm laser and a 528-nm detector
(Aviv Biomedical). Datawere converted from Beckman format, and the
noise-correctedvan Holde–Weischet plots were analyzed using
UltrascanIIIsoftware (5, 6). Boundary fractions above and below the
50%bound point (corresponding to ∼4S for CA hexamer)
directlyyielded bound fractions, which were then used for curve
fitting.Experiments were performed three independent times.
Fluorescence Polarization Spectroscopy. Increasing
concentrationsof CA protein constructs were titrated into 145 nM
and 86 nM offluorescent CPSF6 and NUP153 peptides, respectively.
Titrationswere performed in 50 mM Tris (pH 8) and 50 mM NaCl.
Themeasured polarization was plotted vs. CA protein
concentrationand fitted to a single-site model using Gnuplot.
Experiments wereperformed three independent times.
1. Emsley P, Cowtan K (2004) Coot: Model-building tools for
molecular graphics. ActaCrystallogr D Biol Crystallogr 60(Pt 12 Pt
1):2126–2132.
2. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based
system for macro-molecular structure solution. Acta Crystallogr D
Biol Crystallogr 66(Pt 2):213–221.
3. Yang Y, Luban J, Diaz-Griffero F (2014) The fate of HIV-1
capsid: A biochemical assayfor HIV-1 uncoating. Methods Mol Biol
1087:29–36.
4. Stremlau M, et al. (2006) Specific recognition and
accelerated uncoating of retroviralcapsids by the TRIM5α
restriction factor. Proc Natl Acad Sci USA 103(14):5514–5519.
5. Brookes E, Demeler B, Rosano C, Rocco M (2010) The
implementation of SOMO(SOlution MOdeller) in the UltraScan
analytical ultracentrifugation data analysissuite: Enhanced
capabilities allow the reliable hydrodynamic modeling of
virtuallyany kind of biomacromolecule. Eur Biophys J
39(3):423–435.
6. Demeler B, Brookes E, Wang R, Schirf V, Kim CA (2010)
Characterization of reversibleassociations by sedimentation
velocity with UltraScan. Macromol Biosci 10(7):775–782.
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Fig. S1. Side by side comparison of the NTD–CTD interface in (A)
the current structure bound to PF74, and (B) unbound hexamer.
Fig. S2. Isothermal titration calorimetry analysis of PF74
binding to HIV-1 CA. In all panels: (Upper) Representative
titration data; (Lower) integrated bindingisotherms and fits to a
single-site model. (A) Isolated NTD. (B) Wild-type CA, which
undergoes a monomer-dimer equilibrium in solution. (C) Monomeric
CAW184A/M185A. (D) Disulfide-stabilized A14C/E45C/W184A/M185A CA
hexamer. (E) Disulfide-stabilized N21C/A22C/W184A/M185A CA
pentamer. (F) R173Ahexamer. (G) R173K hexamer. (H) Isolated
CTD.
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Fig. S3. Isothermal titration calorimetry analysis of PF74
binding to HIV-1 CA escape mutants. In all panels: (Upper)
Representative titration data; (Lower)integrated binding isotherms
and fits to a single-site model. (A) T107N NTD. (B) T107N hexamer.
(C) 5Mut NTD. (D) 5Mut hexamer.
Fig. S4. CPSF6 and PF74 both bind to the NTD using a key phenyl
ring. (A) Superposition of the CPSF6-bound and PF74-bound NTD–CTD
interface, with PF74 inwhite and CPSF6 in orange. The R2 moiety of
PF74 and CPSF6 Phe321, which are equivalent in the two structures,
are encircled. (B) View of the phenyl ringsbound to its NTD pocket.
The conserved set of hydrogen bonds to Asn57 is shown as yellow
lines.
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Fig. S5. Raw sequence alignment of CTD sequences from the
curated sequence databases maintained by the Los Alamos National
Laboratory.
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Table S1. Crystallographic statistics
Data collectionLigand PF74 CPSF6Beamline APS 22-BM APS
24-ID-CSpace group P6 P212121Cell dimensions a = b = 91.4, c = 56.6
Å a = 134.8, b = 136.0, c = 207.2 Å
α = β = 90°, γ = 120° α = β = γ = 90°Resolution range, Å 50–2.00
(2.07–2.00) 48.47–2.60 (2.74–2.60)Rsym, % 8.7 (58.5) 6.5 (86.3)Mean
/σ 43.9 (5.3) 20.2 (2.3)Completeness, % 99.9 (100) 100 (100)Average
redundancy 11.1 (11.2) 6.8 (7.1)Average mosaicity, o 1.08Wilson
B-factor, Å2 28.0 62.7
RefinementResolution range 26.6–2.0 (2.1–2.0) 48.47–2.60
(2.67–2.60)No. of unique reflections 17,520 (2,875) 117,427
(8,192)Reflections in free set 897 (161) 2,005 (144)Rwork, % 22.0
(20.7) 21.9 (29.5)Rfree, % 27.1 (26.5) 25.9 (35.3)No. of
nonhydrogen atoms
Protein 1,662 19,802Solvent 100 —Ligand 32 1,110
Average B-factor, Å2
Protein 33.8 73.0Solvent 37.5 —Ligand 26.0 91.2
Coordinate deviationsBond lengths, Å 0.011 0.003Bond angles, o
1.135 0.564
Ramachandran plotFavorable 0.99 0.97Allowed 0.01 0.02Disallowed
0 0.01
PDB ID 4QNB 4WYM
Values in parentheses are for the highest-resolution shell.
Table S2. Summary of isothermal titration calorimetry analysis
of PF74 binding to HIV-1 CA
CA construct Mutations tested Oligomeric state Kd (μM)* ΔH
(Cal/mol)* ΔS (kCal·mol–1·deg–1)* N*,†
CA None Monomer/dimer 3.70 ± 1.13 −9,467 −6.5 1.14NTD None
Monomer 3.58 ± 0.78 −12,983 −18.1 1.13CTD None Monomer/dimer No
bindingCA None Monomer 4.26 ± 1.21 −12,200 −15.9 1.01NTD T107N
Monomer 3.62 ± 0.08 −13,800 −20.9 1.18NTD 5Mut Monomer 7.67 ± 0.12
−13,966 −23.0 1.03CA None Hexamer 0.26 ± 0.09 −10,003 −3.7 0.99CA
T107N Hexamer 0.29 ± 0.03 −10,300 −4.3 1.13CA 5Mut Hexamer 1.15 ±
0.11 −7,557 2.1 1.03CA R173A Hexamer 2.01 ± 0.27 −7,928 −0.25
1.21CA R173K Hexamer 0.51 ± 0.07 −8,014 1.8 1.02CA None Pentamer
0.28 ± 0.01 −12,136 −10.3 0.99
*In all cases three separate ITC runs were
averaged.†Stoichiometry of ligand:protein interaction in terms of
monomers of CA protein (SI Materials and Methods).
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Table S3. Summary of binding data for fCPSF6 and fNUP153
Peptide CA construct Mutations tested Oligomeric state Kd (μM)*
Technique
fCPSF6 NTD None Monomer 1,170 AUCfCPSF6 CA None Monomer/dimer
Not determined AUCfCPSF6 CA None Hexamer 99.8 AUCfCPSF6 NTD None
Monomer 871.7 FPfCPSF6 CA None Monomer/dimer 436 FPfCPSF6 CA None
Hexamer 83.3 FPfCPSF6 CA K182A Hexamer 423.8 FPfCPSF6 CA K182R
Hexamer 587.5 FPfNUP153 NTD None Monomer 3,369 FPfNUP153 CA None
Hexamer 484 FP
*In all cases three independent measurements were averaged. AUC,
analytical ultracentrifugation; FP, fluores-cence polarization.
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