Fig 1.
Changes in viral morphology resulting from different purification methods.
Contrast inverted cryo-ET central slices of clarified supernant fluid collected directly from infected Vero cells and either imaged after being flash frozen (A, B), fresh (C, D), or ultracentrifuged (E-G). Inserts represent enlarged regions of the viral glycoprotein layer. (H) Viral particle aspect ratio of all three purification methods (n = 50). Scale bars: (A-G) 50 nm.
Fig 2.
Viral tomographic reconstructions.
(A) Contrast inverted cryo-ET central slice of HPIV3 before receptor engagement. Red arrows indicate ribonucleoprotein helical tubes. (B, C) Enlarged regions of the surface glycoproteins with HN and F in tight arrangement. (D, E) Central (D) X slice and (E) Y slice through the subtomogram average of HPIV3 surface glycoproteins. (F, G) Sub-volume average of surface glycoproteins with crystal structure of the HN dimer (PDB ID: 4MZA) and the cryo-EM structure of pre-fusion F (PDB ID: 6MJZ) in green and pink (respectively), fitted into the sub-volume average. (H) Top-down view of HN and (I) top-down view of F. Scale bars: (A) 50 nm and (D, E) 10 nm.
Fig 3.
Imaging the interaction of the HPIV3 virions with a natural host receptor.
To capture viral-host interactions and prevent the activation of F, samples were incubated at 4°C prior to vitrification. (A, B, C) Contrast-inverted (A) cryo-ET and (B, C) cryo-EM images of HPIV3 interactions with target erythrocyte fragment membranes. Insets: enlarged areas show thin lines of density connecting HN to the target erythrocyte fragment membranes. (D, E) Enlarged regions (D) distal and (E) proximal to the viral-host interaction site with accompanying density line plots of each region. Relative density measurements in arbitrary units (a.u.) of the space between the viral envelope (V.E.) and HN or the target human erythrocyte fragment membrane (H.E.) with distances measured from positions of half-maximum density (distance at half-maximum density of outer leaflet to peak intensity distal to membrane), showing the positions of each viral envelope glycoprotein. (F) Box plot of distances measured from regions equivalent to (D) and (E) with each distance represented by boxes of the same color as those overlaid in the graphs of panels (D) and (E). Dashed vertical lines indicate half maximum distance of the viral envelope (V.E.), HN, and host envelope (H.E.). Numbers indicate average heights of 38 measurements with +/- standard deviations. Scale bars: (A, B, C) 50 nm.
Fig 4.
Capture of the transient intermediate state of F with lipid-conjugated fusion inhibitory peptides.
To capture the transient intermediate fusion state just after HN activates F, receptor-bearing target erythrocyte fragment membranes were exposed to virus on grids at 37°C in the presence of lipid conjugated fusion inhibitory peptides (i.e., VIKI-PEG4-chol), prior to vitrification. (A) Schematic of lipid-conjugated peptides inserting into the target cell membrane via their lipid tails and “locking” the extended F in its transient intermediate state, preventing refolding to the post-fusion conformation. (B, D) Contrast-inverted images where viral particles can be observed attached to target erythrocyte fragment membranes using (B) cryo-EM and (D) cryo-ET. (C, E) Enlarged region of interactions between the viral and target erythrocyte fragment membranes where elongated densities linking both membranes are visible. Insets include cyan lines where distance plot measurements were taken. (C,D,E, bottom) Density line plots revealing a repeating 20–35 Å-wide density. Scale bars: (B, E) 50 nm.
Fig 5.
Fusion of HPIV3 with a target erythrocyte fragment membrane.
(A, B) Contrast-inverted viruses that underwent fusion with target erythrocyte fragment membranes (top) with density color representation overlaid below. The purple color represents the dense viral ribonucleoprotein, and the yellow color represents the erythrocyte content. (C) Negative control where grids were kept at 4°C, prior to vitrification to prevent fusion of target erythrocyte membranes with the viruses. (D) Particle aspect ratio of viruses in the presence of zanamivir (also incubated at 4°C, as in C), compared to fused particles (n = 23). (E) Fusion with a small target erythrocyte fragment membrane reveals a lack of prefusion F density near the sites of fusion (inserts). (F) A possible instance of hemifusion where the target erythrocyte fragment membrane surface shows no evidence of surface glycoproteins, and the viral surface shows a lack of prefusion F density near the sites of fusion (inserts). Scale bars: (A-C) and (E, F) 50 nm.
Fig 6.
Sequence of events in HPIV3 entry, corresponding to cryo-electron microscopy imaging.
(A) HN (green) and F (dark pink) can be found densely packed on the viral surface (light pink) (Image from Fig 1A). (B) Sialic acid (purple) binding to HN occurs in the presence of a host target membrane (blue) (Image from Fig 3B). (C) Upon triggering of F by HN, F undergoes a large conformational change from a pre-fusion globular structure to an extended structure that crosses both membranes (Image from Fig 4B). (D) After this intermediate state, F folds back onto itself, pulling both membranes towards each other, creating a pore in a process that ultimately results in a merged membrane. Scale bars: (A-D) 50 nm.