Figure 1.
Functional purification of assembly-competent HIV-1 capsid proteins.
(A) Flow diagram describes the functional purification scheme. Addition of NaCl to 2.5 M final concentration to the ammonium sulfate-saturated capsid solution and subsequent dissolution of the capsid-enriched pellet permits recovery of highly pure HIV-1 CA proteins after passage through the anion-exchange chromatography step. (B) SDS-PAGE analysis of fractions obtained during purification of wild-type capsid protein from E. coli lysate. The gel was stained with SimplyBlue™ Safe Stain. Lanes: M, SeeBlue Plus2 marker (the molecular weights of standards are depicted on the left-hand side of the gel); 1, total extract; 2, inclusion bodies; 3, soluble fraction; 4, supernatant after ammonium sulfate cut; 5, resolubilized 25% ammonium sulfate cut pellet; 6, unpolymerized fraction after 1st addition to 2.5 M NaCl; 7, solubilized protein pellet fraction after first polymerization; 8, unpolymerized fraction after 2nd addition of NaCl to 2.5 M final concentration; 9, solubilized protein pellet fraction after second polymerization; 10, insoluble pellet after dissolution of the polymerized capsid pellet from second round of polymerization; 11, Anion exchange chromatography flow-through of CA. Identity of the purified protein is confirmed by Time of Flight Mass Spectrometry (Figure S4).
Table 1.
Process summary of WT CA purification by polymerization.
Figure 2.
Solution oligomeric state of functionally purified WT CA and assembled CA 4Mu hexamer.
(A) Full-length wild-type capsid protein (WT CA) and (B) hexameric capsid construct obtained by disulfide cross-linking of CA 4Mu monomer ([A14C,E45C,W184A,M185A]CA). Sedimentation velocity experiments were performed in 50 mM sodium phosphate buffer (pH 7.5) at 25°C and 45,000 rpm. Absorbance scans at 280 nm were collected for CA FL (A) at 0.94 µM, 1.88 µM, 3.75 µM, and 7.5 µM, and for CA 4Mu Hexamer (B) at 4.13 µM, 8.25 µM (±1 mM TCEP) and 16.5 µM (expressed as monomers of CA 4Mu). Sedimentation coefficient distribution – c(s) - was calculated using Sedfit program. WT CA contains monomers in equilibrium with dimers, whereas CA 4Mu hexamer preparation contains 94% of 4Mu monomers in hexameric form and 6% as a mixture of monomers, dimers and trimers. Treatment with 1 mM TCEP converts hexamers back to 4Mu CA monomers.
Figure 3.
Polymerization kinetics of (A) full-length wild type capsid (WT CA) and (C) mutant capsid [A14C,E45C,W184A,M185A] monomer (CA 4Mu).
Assembly reactions were induced by 2-fold dilution of capsid protein into buffer containing 50 mM sodium phosphate (pH 7.5), 4 M NaCl at 25°C. The increase in samples turbidity (absorbance at 350 nm) due to the formation of large oligomeric structures was plotted as a function of time. Final concentration of capsid protein in reaction is depicted on the right side of the kinetic traces. The data points represent an average of three determinations with standard deviation shown as grey bars. The dependence of the initial rate of capsid polymerization is plotted against the concentration of (B) WT CA and (D) CA 4Mu monomer. The initial rate was calculated from the slope of the linear phase of the polymerization curve.
Figure 4.
Transmission electron microscopy analysis of structures present in the solution of (A) polymerized WT CA and (B) assembled CA 4Mu Hexamer.
Objects were deposited onto the grid and visualized by the negative staining. Black bar on micrographs represents scale in nm. (A) Tube-like and cone-like structures can be observed in solution of WT CA polymerized at 40 µM concentration in buffer containing 50 mM sodium phosphate (pH 7.5), 2 M NaCl, 0.005% Antifoam 204. (B) Hexameric assemblies are clearly visible in a solution containing 19.5 µM of disulfide cross-linked CA 4Mu (expressed as CA monomers concentration) in 20 mM Tris (pH 8.0), 10% glycerol buffer.
Figure 5.
Inhibition of full-length wild-type capsid polymerization kinetics by two CA binding agents.
(A, B) CAI-4 is a peptide that binds to the C-terminal domain of CA. (C, D) BM2 is a small molecule inhibitor described by Boehringer Ingelheim that binds to the CA N-terminal domain. WT CA was pre-incubated with each inhibitor. Assembly reactions were induced by 2-fold dilution of capsid protein into buffer containing 50 mM sodium phosphate pH 7.5, 4 M NaCl at 25°C. The increase of turbidity (absorbance at 350 nm) of 40 µM solution of WT CA in the presence of increasing concentrations of inhibitor (depicted on the right side of kinetic traces) was plotted as a function of time. The data points represent an average of three determinations with standard deviation shown as grey bars. The dose response curves of inhibition of polymerization kinetics and the concentration of inhibitor resulting in a half-maximal inhibition (IC50) for (B) CAI-4 and (D) BM2. The percent of inhibitor activity (% Activity) represents the percent of decrease of the initial rate of polymerization reaction.
Figure 6.
Binding of CAI peptide and BM2 to WT CA and CA 4Mu Hexamer analyzed by Surface Plasmon Resonance.
(A) Sensorgram data of CAI binding to WT CA. CAI was injected at the highest concentration of 100 µM and ten 3-fold serial dilutions thereof. Each trace was collected in duplicate. (B) Fit of sensorgram data in A by fitting the equilibrium response at each concentration to a simple 1∶1 binding isotherm model. (C) Sensorgram data of CAI binding to hexamer. CAI concentrations used were the same as in A. (D) Attempt to fit sensorgram data in C to a simple 1∶1 binding isotherm model. Due to insufficient response, an accurate equilibrium dissociation constant for this interaction could not be obtained. The KD for this interaction was simply cited as >100 µM. (E) Sensorgram data for the binding of BM2 to WT CA (black curve, sensorgram: red curve kinetic analysis using a simple 1∶1 kinetic binding model). The KD for this interaction was calculated from the ratio of the kon and koff rate constants obtained from this fit. (F) Sensorgram data for the binding of BM2 to CA 4Mu Hexamer. The affinity of BM2 to hexamer is too weak to allow for quantification of the kon and koff rate constants.