Structure and function of a genetically engineered mimic of a nonenveloped virus entry intermediate

Abstract

Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family Nodaviridae, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membrane-disrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.

FIG. 1.

Calcium-binding sites in Flock House virus. (A) Locations of calcium-binding sites in one icosahedral asymmetric unit (iASU) of the FHV capsid. The A, B, and C subunits in the iASU are colored blue, red, and green, respectively, with the gamma peptides shown in yellow and bound calcium ions colored magenta. The view on the top is from the outside of the virus, while that on the bottom is from the interior of the virus. The positions of the metal ions at the interfaces of subunits are indicated by black arrows. (B) Close-up stereoscopic views of the residues involved in calcium chelation at the quasi-3-fold axes of symmetry (B) and the interface of A and B subunits (C). The distance between each residue and calcium ion is given in angstroms.

FIG. 2.

Calcium site mutations do not affect the assembly and maturation of FHV. (A) Amino acid changes in three calcium site mutants (expressed as virus or virus-like particles) and the maturation-defective mutant (expressed as virus) of FHV. (B) SDS-PAGE of purified virus particles produced in Drosophila DL-1 cells transfected with FHV RNA1 (which codes for FHV polymerase) along with either wild-type RNA2 (which codes for capsid protein α) or altered RNA2 corresponding to FHV^CM1, FHV^CM2, FHV^CM3, or FHV^MD. Each lane represents an equivalent volume of purified particles, and the positions of α and the maturation cleavage product β are indicated. Virus particles were purified from cells 36 h posttransfection; thus, they were produced from multiple cycles of replication. (C) SDS-PAGE of purified virus-like particles (VLPs) containing either the wild-type or a calcium-site-mutated capsid protein sequence. Each lane represents an equivalent volume of purified protein, and the positions of α and β are indicated. (D) Electron micrographs of wild-type and FHV^CM3 VLPs, stained with 2% Nano-W.

FIG. 3.

Calcium site mutations affect the stability of VLPs. Results of differential scanning calorimetry of wild-type (solid line), FHV^CM1 (open squares), FHV^CM2 (filled circles), and FHV^CM3 (open triangles) VLPs, with each VLP at a concentration between 0.2 and 0.4 mg/ml in 50 mM HEPES (pH 7.0), are shown. The Cp (kilocalories of heat absorbed per mole per °C) of the sample is plotted as a function of temperature. The curves for the buffer solution only were subtracted from the sample curves in each case. The temperatures for the major endothermal transitions are given. The traces are representative data from three separate experiments.

FIG. 4.

Calcium removal decreases the stability of the FHV capsid. Results of differential scanning calorimetry of wild-type FHV in 50 mM HEPES (pH 7.0) (solid line) and wild-type FHV dialyzed against a 50 mM HEPES-5 mM EGTA solution (open circles), both at a concentration of 0.3 mg/ml, are shown. The traces are representative data from three separate experiments.

FIG. 5.

Calcium-site-mutated VLPs bind to DL-1 cells but cannot rescue the infectivity of maturation-defective FHV. (A) Progeny virus produced by coinfecting 1 × 10⁸ Drosophila DL-1 cells with 1.5 × 10³ particles/cell of D75N/N363T FHV and 9 × 10³ particles/cell of wild-type VLPs or each of the mutated VLPs. [³⁵S]methionine-cysteine-labeled progeny virus was quantified, with the amount of progeny produced during coinfection with D75N/N363T FHV and wild-type VLPs normalized at 100%. (B) FACS analysis of the binding of wild-type or mutated VLPs to 1 × 10⁶ Drosophila DL-1 cells for 1 h at 4°C. Bound particles were detected using a rabbit polyclonal primary antibody against FHV capsid protein and a goat anti-rabbit secondary antibody conjugated with Alexa 488 (Molecular Probes). Five percent BSA was added to cells as a negative control, followed by antibody incubations. Data shown are representative of two separate experiments.

FIG. 6.

The gamma peptide is more exposed in calcium site mutants. (A) Relative amounts of sulforhodamine B released from DOPC liposomes after 15 min of incubation with 6.37 × 10¹¹ particles of wild-type FHV (circles) or FHV^CM3 (squares) VLPs, at different pHs. The level of fluorescence dequenching achieved upon the addition of 0.1% Triton X-100 (TX100) to DOPC liposomes is considered 100%. (B) Relative amount of sulforhodamine B released from liposomes after 1 h of incubation with 6.37 × 10¹¹ particles of each virus or VLP as indicated. The dye release by 0.1% TX100 from DOPC liposomes is considered 100%. (C) MALDI-TOF mass analysis of a time course proteolysis reaction comparing wild-type and FHV^CM3 VLPs. Peptides are released more rapidly from the mutant particles (series on the right), indicating that they are more dynamic. In this reaction the enzyme-to-FHV protein ratio was 1:4,000 (wt/wt). Reactions using more enzyme with the wild-type particles confirmed that the cleavage pattern was the same.

FIG. 7.

Crystal structure of the FHV^CM3 VLP. (A) Density corresponding to the calcium-binding site at the quasi-3-fold axes of the FHV^CM3 VLP (dark gray mesh), with the crystal structures for FHV^CM3 VLP and wild-type FHV (including the calcium ion) built in. The A, B, and C subunits of the FHV^CM3 VLP are shown in blue, red, and green, respectively. The subunits of wild-type FHV are in gray, and the calcium ion present in the quasi-3-fold axes of wild-type FHV is shown as a magenta sphere. (B) Density corresponding to the amphipathic gamma helices (residues 364 to 381) in subunits A, B, and C of the FHV^CM3 VLP, with the wild-type FHV crystal structure built in. The A, B, and C subunits of wild-type FHV are shown in blue, red, and green, respectively. Density for the gamma peptides is mostly disordered in subunits A and B of the FHV^CM3 VLP.