Fig 1.
(a) The coding scheme of Orsay δ and CP-δ fusion protein. CP-δ, which is produced by ribosomal frameshifting, is comprised of the full-length CP, a 29-aa linker, and the full-length δ. The Orsay CP can be divided into three parts, the N-terminal peptide, shell, and the spike domain. The five δ and CP-δ truncation mutants are shown below. (b) TEM images of δ by negative staining. On the right are five enlarged δ aligned in the horizontal direction. Scale bar, 500 Å. (c) CD spectra of δ. The experimental data and the fitting curves are shown in red and blue, respectively. The table below compares the secondary structure contents calculated from CD and sequence-based prediction. (d) δ domain map. There is a slightly enlarged tail domain at the N-terminus (aa1-66), a small globular domain in the middle, and a large globular head at the C-terminus.
Fig 2.
Truncation mutants of δ and CP-δ.
(a) SDS-PAGE showing five purified proteins. (b) Negative-staining EM images. δ(1–241), δ(1–162) and the midi-fusion all formed fiber-shaped molecules. Some of these fibers are highlighted by arrows. Scale bar, 500 Å. (c) Length measurements for the two δ truncation mutants in comparison with the full-length δ. (d) Length measurements for the midi-fusion protein in comparison with δ(1–162).
Fig 3.
(a) Ribbon diagram. The five subunits are shown in different colors. (b) Surface representation colored by electrostatic potential. Both a side-view (left) and a top view (right) are provided in (a) and (b). (c) Secondary structure for the five chains in a pentamer. Bold letters highlight the residues with their side chains pointing to the interior of the helical bundle. α-helices are shown by boxes, non-structured loops are represented by black lines, and disordered regions are shown by dotted lines. (d) An electron density map showing water-like features inside the helical bundle.
Table 1.
Crystallographic statistics.
Fig 4.
Full-length δ forms exclusive pentamers.
(a) Mass spectrometry confirms the predominance of the homo-pentameric complex. The experimental mass of the intact complex is 192214.8 ±1 Da and is almost exactly the mass expected for a homo-pentamer. There is no indication in the mass spectrum of the existence of an alternative stoichiometry, including monomer. Note that due to the natural variance in isotopic composition the average mass of a protein complex of this size is inherently uncertain. The sum of the uncertainties of the relative atomic masses for this composition results in an estimate of the natural variation of approximately ±9Da, indicating that the observed difference of approximately 4 Da is well within the range expected. (b) Molar mass analysis by analytical ultracentrifugation. The integrated molar mass of the major peak is consistent with a pentameric species. Higher molecular weight species are also visible, but are present at lower concentration. Molecular species > 800,000 Da are not shown for clarity. Minor peaks may explained by modeling inaccuracies and/or contaminating proteins.
Table 2.
Integration results from the PCSA-DS.
Fig 5.
CP-δ forms particle associated fibers.
(a) EM images of Orsay VLPs containing CP-δ. Protruding fibers are highlighted by orange stars. Scale bar, 500 Å. (b) Length measurements of particle-associated fibers in comparison with the full-length δ fibers. (c) SDS-PAGE of the Orsay VLP. Western blot was performed using an anti-His antibody. (d) An Orsay capsid model with CP-δ fibers situated at the five-folds. On five-fold vertices, the CP portion and the δ portion of the CP-δ are shown in light green and orange respectively. The rest of the CP molecules in the capsid are shown in dark green. The Orsay, ~350-Å in diameter, and the CP-δ fiber, ~400-Å in length, are approximately drawn to scale.
Fig 6.
Site-directed mutagenesis and infectivity assays.
(a) K43 forming a salt bridge with D45. Only a portion of the structure is shown. (b) L44 facing the hydrophobic interior of the helical bundle. (c) Transparency test for wild-type (WT) and mutant Orsay viruses. As displayed in the inset, Orsay-infected worms, but not the control, show a transparent intestine particularly at the anterior region as indicated by red brackets. Scale bar, 100μm. (d) Viral load of wild-type and mutant viruses measured by qRT-PCR. In (c-d), two independent transgenic lines for each mutant virus were tested. ATG represents the δ-null mutant. Three independent trials (biological replicates) were conducted. Error bar, standard error from three biological replicates. *, p<0.05; ***, p<0.001 compared with WT using Student’s t-test. (e) A typical viral titer determination result. The arrow indicates the viral concentration used for the protein-competition assay. (f) Adding full-length δ in the culture medium reduces viral infectivity. Delta-N represents the δ(1–101) deletion mutant. Bars and error bars show mean and standard deviation from three independent trials. 12 plates were tested in each trial for each protein/condition. *, p<0.05, Student’s t-test, paired samples.