Fig 1.
Analysis of PFV infecting HT1080 cells.
A, B- Low magnification view of PFV infected cells showing budding at the plasma membrane (indicated by * symbol) (A) and viruses in intracellular vacuoles (symbol V) (B). Free (non-enveloped) cytoplasmic capsids can be seen as well (labeled Cap in (B)). C, D- Higher magnification view of viruses budding at the plasma membrane (C) and viruses possibly budding in vacuole (D–zoom in the rectangular region of (B)). E- 0.8 nm thick tomogram slice through five different viruses budding at the plasma membrane. The red arrowheads point to the intermediate shell between the capsid and the plasma membrane. Scale bars in A—B, C—D and E are 500, 200 and 60 nm respectively.
Fig 2.
Cryo-electron tomography of wt PFV.
Central slices through wt PFV virions having a complete capsid (Class A), an incomplete capsid (Class B), no capsid (Class C) or miscellaneous particles (Class D). For each class, one virus has its capsid, intermediate shell, viral membrane and glycoproteins colored in red, green, blue and yellow respectively. The interior of Class C virus having no characteristic features is left uncolored. For each class A virus, the corresponding radial density profile is shown and the various peaks corresponding to the glycoprotein (G), viral membrane (M), intermediate shell (I) and capsid (C) are labeled on one plot. Scale bar is 60 nm.
Fig 3.
Ultrastructure of PFV virus interior.
A- Central slice through a wt PFV virus imaged by cryo-ET. The white and red arrows point to some of the interactions/connections between capsid / intermediate shell and intermediate shell / viral membrane respectively. The blue parenthesis highlights local organization in the intermediate shell. B- Same as (A) except that this virus has no glycoprotein sticking out of the membrane but it still has a capsid with an intermediate shell (colored in green). C- Tomogram section of PFV iNAB mutant showing viral particles without any internal ordered structure. Black arrows point internal densities located close to the viral membrane but distinct from the intermediate shell of wt and iFuse PFV. D- 2D classification on the capsid alone of iFuse mutant viruses imaged by cryo-EM. Five 2D class averages are shown which could be interpreted as 2D projections of a pseudo-icosahedral object viewed along the 5, 5, 3, 2 and 5-fold symmetry axes respectively. Scale bars are 60 nm for each panel.
Fig 4.
Subtomogram averaging of PFV glycoprotein.
A–C- 0.8 nm thick tomographic slice perpendicular to the glycoprotein long axis from the wt, iNAB and iFuse mutants and its corresponding schematic of interlocked hexagonal assemblies of trimers. Numbers are indicated at the center of each hexagon and triangles represent the position of each trimer of Env in the hexagonal network. The wt virus shows ordered region of glycoprotein (represented on the corresponding schematic on the right) next to less ordered one while both mutants have more ordered hexagonal networks. D–I- Subtomogram averaging on glycoproteins of wt (top), iNAB (middle) and iFuse (bottom) mutants. D–F and G–I show a side view of a single trimer and a top view of intertwined hexagonal assemblies respectively. The two arrows in (G) point to less well defined peripheral trimers on wt particles. Scale bars are 100 Å.
Fig 5.
In Situ single particle 3D reconstruction of PFV glycoprotein by cryo-EM.
A- Side view of the sharpened 3D reconstruction of an hexagonal assembly of trimeric glycoproteins from the iFuse mutant (6-fold symmetry applied, ~10 Å resolution at FSC = 0.143). Spikes are colored alternately yellow and salmon, viral membrane is gray. B- Gray scale section at the level of the dotted line in (A). The triangle and hexagon symbols indicate the position of each trimer and of the 6-fold axis orthogonal to the section plane respectively. The two white arrowheads delimit the region where the trimers are interacting with each other. C, D- Side views (full (C) and cut-away (D) views) of a single PFV Env trimer (sharpened map) after 3-fold symmetry application (~9 Å resolution at FSC = 0.143). The densities corresponding to the extracellular domains and the viral membrane are colored salmon and gray respectively in C. The three central helices attributed to gp48 fusion peptide are represented by three green α helices of 22 residues long each. The TMHs are represented by three inner (colored blue) and three outer (colored orange) α helices. In D, the densities surrounding the three central helices and the three inner and outer TMHs are colored green, blue and orange respectively while the remaining of the spike is gray colored. E, F and G- Grayscale sections through the reconstruction shown in (C): E- central side view as in (D), F and G- top views at the level of the dotted lines in (E) through the three helix bundle and the TMHs respectively (the three outer and inner TMHs are labeled 1, 2, 3 and 4, 5, 6 respectively). Scale bars are 50 Å in A, B, D, E.