Characterization of the In Vitro HIV-1 Capsid Assembly Pathway

During the morphogenesis of mature human immunodeficiency virus-1 cores, viral capsid proteins assemble conical or tubular shells around viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which capsid proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period, followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle preexisting tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature human immunodeficiency virus-1 core assembly and avenues for antiviral inhibition.     

Quantitative Determination of Site-Specific Conformational Distributions in an Unfolded Protein by Solid-State Nuclear Magnetic Resonance

Solid-state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid-state ¹³C NMR spectra of samples prepared with uniform ¹³C labeling of selected residues show relatively sharp cross-peaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most cross-peaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid-state NMR techniques that provide independent constraints on backbone ? and ψ torsion angles in samples with sequential pairs of carbonyl ¹³C labels. Without GdnHCl, the combined data are well fit by α-helical conformations. At [GdnHCl] = 4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of α-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl] = 7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid-state NMR.     

Disulfide Bond Stabilization of the Hexameric Capsomer of Human Immunodeficiency Virus

The human immunodeficiency virus type 1 capsid is modeled as a fullerene cone that is composed of ∼ 250 hexamers and 12 pentamers of the viral CA protein. Structures of CA hexamers have been difficult to obtain because the hexamer-stabilizing interactions are inherently weak, and CA tends to spontaneously assemble into capsid-like particles. Here, we describe a two-step biochemical strategy to obtain soluble CA hexamers for crystallization. First, the hexamer was stabilized by engineering disulfide cross-links (either A14C/E45C or A42C/T54C) between the N-terminal domains of adjacent subunits. Second, the cross-linked hexamers were prevented from polymerizing further into hyperstable capsid-like structures by mutations (W184A and M185A) that interfered with dimeric association between the C-terminal domains that link adjacent hexamers. The structures of two different cross-linked CA hexamers were nearly identical, and we combined the non-mutated portions of the structures to generate an atomic resolution model for the native hexamer. This hybrid approach for structure determination should be applicable to other viral capsomers and protein-protein complexes in general.     

Conformation of the HIV-1 Gag protein in solution

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.     

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

Partial site-specific assignment of a uniformly C, N enriched membrane protein, light-harvesting complex 1 (LH1), by solid state NMR

Partial site-specific assignments are reported for the solid state NMR spectra of light-harvesting complex 1, a 160 kDa integral membrane protein. The assignments were derived from 600 MHz ¹⁵N-¹³CO-¹³Cα and ¹⁵N-¹³Cα-¹³CX correlation spectra, using uniformly ¹³C, ¹⁵N enriched hydrated material, in an intact and precipitated form. Sequential assignments were verified using characteristic ¹⁵N-¹³Cα-¹³Cβ side chain chemical shifts observed in 3D experiments. Tertiary contacts found in 2D DARR spectra of the selectively ¹³C enriched sample provided further confirmatory evidence for the assignments. The assignments include the region of the Histidine ligands binding the Bacteriochlorophyll chromophore. The chemical shifts of Cα and Cβ resonances indicated the presence of typical α-helical secondary structure, consistent with previous studies.