Higher-order structure of the Rous sarcoma virus SP assembly domain

Abstract

Purified retroviral Gag proteins can assemble in vitro to form immature virus-like particles (VLPs). By cryoelectron tomography, Rous sarcoma virus VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. We hypothesize that the structure represented by these densities is formed by amino acid residues immediately downstream of the folded CA, namely, the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and we propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, we have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues near the N and C termini, as well as three hydrophilic residues within SPA. We interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag compared that of assembled VLPs showed strong protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration-dependent manner. Analytical ultracentrifugation showed concentration-dependent self-association of the peptide into a hexamer. Taken together, these results provide strong evidence for the formation of a critical six-helix bundle in Gag assembly.

Importance: The structure of a retrovirus like HIV is created by several thousand molecules of the viral Gag protein, which assemble to form the known hexagonal protein lattice in the virus particle. How the Gag proteins pack together in the lattice is incompletely understood. A short segment of Gag known to be critical for proper assembly has been hypothesized to form a six-helix bundle, which may be the nucleating event that leads to lattice formation. The experiments reported here, using the avian Rous sarcoma virus as a model system, further define the nature of this segment of Gag, show that it is in a higher-order structure in the virus particle, and provide the first direct evidence that it forms a six-helix bundle in retrovirus assembly. Such knowledge may provide underpinnings for the development of antiretroviral drugs that interfere with virus assembly.

FIG 1

RSV SP predicted helix and point mutations. (A) Schematic diagram of the RSV SP predicted helical sequence. Each residue within the predicted helix was mutated to alanine or serine. All mutations were made within ΔMBDΔPR, a truncated version of RSV Gag that contains a deletion of the membrane-binding domain (MBD) of MA and of the PR domain. (B to D) Examples of observed in vitro assembly using negative-stain EM. ΔMBDΔPR, wild-type protein (control); A₄₇₈S, assembly-negative mutant; M₄₈₈A, assembly-positive mutant. Scale bars are 500 nm.

FIG 2

Model of the RSV SPA amphipathic helix and putative six-helix bundle. (A) A helical wheel representation of the predicted SP helix (enumerated according to each residue's position within full-length RSV Gag). Polar, uncharged residues are green, negatively charged residues are pink, and nonpolar/hydrophobic residues are yellow. Residues indicated by asterisks can be mutated without abrogating in vitro assembly. (B) The proposed organization of the putative six-helix bundle. The Roman numerals I and II designate opposite but putatively interacting sides of the large hydrophobic face.

FIG 3

Hydrogen-deuterium exchange analysis of unassembled and assembled ΔMBDΔPR. (A) SP predicted helix sequence. The two peptic fragments corresponding to regions in RSV SP are underlined. Vertical lines correspond to natural protease cleavage sites in Gag. (B) Mass-to-charge spectrum for the peptide of residues 477 to 488 in unassembled ΔMBDΔPR protein (blue) and assembled VLP (red). (C) Plot of deuterium incorporation in the peptide of residues 482 to 488. (D) Plot of deuterium incorporation in the peptide of residues 477 to 488 at different time points.

FIG 4

Circular dichroism spectroscopy of SPApep. (A) Amino acid sequence of SPApep. Residues are numbered based on their position in full-length RSV Gag. The exogenous C-terminal cysteine is shown in red. (B) Spectra of SPApep in PBS at various concentrations, measured at 25°C. A comparison of the spectra of SPApep and SPAmut at the highest concentrations, measured at 37°C, is shown in the inset. (C) Quantification of the percent helicity of each spectrum shown in panel B. (D) Spectra of SPApep at 0.20 mM concentration with increasing concentrations of TFE. (E) Quantification of the percent helicity of each spectrum shown in panel D. (F) Spectra of SPApep at 0.033 mM with increasing concentrations of TFE. (G) Quantification of the percent helicity of each spectrum shown in panel F.

FIG 5

Analytical ultracentrifugation. (A) c(s) distributions of SPApep at 1, 5, and 10 mg/ml loading concentrations. An increasing weight-average sedimentation coefficient with increasing concentration is consistent with SPApep existing in rapid equilibrium. (B) Two-dimensional c(s,M) fit for the 10 mg/ml loading concentration SV data. The peak s-value was found at 1.65 S, consistent with the one-dimensional fit.

FIG 6

Model of the putative SPA 6HB. (A) The SPApep 6HB (green ribbon) was modeled using Swiss PDB Viewer. The model was built based on structural alignments with the published crystal structure of a synthetic 6HB, CC_hex (pink ribbon). Putative interacting residues are shown in stick format and are colored according to amino acid type (yellow, hydrophobic; red, negatively charged; blue, positively charged). A seam of hydrophobic residues lines the interface between two adjacent helices. There is also a predicted salt bridge between residues R₄₉₃ and E₄₉₄. (B) Top-down view of the putative SPA 6HB (green ribbon) highlighting potential interacting residues. The top-down view of CC_hex is shown for comparison.

FIG 7

Conserved 12th helix spans the cleavage site between CA and the downstream domain. Sequences show Gag from the last helix of CA_CTD to the first cysteine in the first zinc finger of NC. The sequences are aligned with one another at the end of the 11th helix of CA (blue cylinder). The putative 12th helix of CA is shown as a red cylinder and is found in all retroviral genera except epsilonretroviruses. Protease cleavage sites (both predicted and known) are indicated by vertical lines.