Figure 1.
Transport of HSV1 capsids in neurons during egress.
(A) Bright field image of a hippocampal neuron grown on a holey carbon support film for 7 days. A neurite (white arrow) and cell body (asterisk) are indicated. Bar: 6 µm. (B) Series of time-lapse wide-field fluorescence images of a mid-axon region of a HSV1(KOS)-GFPVP26 infected neuron at 16 hours p.i.. Pictures were taken every 2 seconds, as indicated by the number in the lower left corner. The left image shows an overlay of the fluorescence channel with the bright field image. Arrows indicate the positions of individual viral particles. Bar: 5 µm. (C) Cryo electron microscopy (projection image) of an intact axon at 16 h p.i.. Cytosolic C-capsids are framed in black and cytosolic A-capsids in black dashed squares. (D–F) Slices through the reconstructed tomographic volume obtained from the area of interest in (C). pm: plasma membrane; mt: mitochondria; black arrows: microtubules. Bars in (C–F): 200 nm.
Figure 2.
Directionality and run lengths of intracellular transported viral particles at 16 h p.i..
Each bar corresponds to an individual single run, 15 in total. Particles that entered or ran out from the field of view during the 10 min observation period were also taken into account. These data are coming from two different observations. For more detail see Table S1. Black bars: anterograde transport, white bars: retrograde transport.
Table 1.
Frequency of viral particle types found in middle regions of axons for the HSV1 strains used for infection.
Figure 3.
Subvolume averaging of cytosolic capsids.
Results from subtomogram averaging are presented for (Ai-iv) 41 cytosolic C-capsids, (Bi-iv) 26 cytosolic A-/B-capsids, (Ci-iv) 143 nuclear C-capsids and (Di-iv) 158 nuclear A-capsids. Row (i): central cross sections of the averages. Row (ii): close-up view of the top vertex in row (i). Row (iii): isosurface representation of the averages with a threshold of 1.5σ above the mean density. Row (iv): close-up view of a vertex from row (iii).
Figure 4.
Difference maps between cytosolic capsids and nuclear capsids.
(A) Difference map between cytosolic C-capsids and nuclear C-capsids, superimposed onto the nuclear C-capsids average. (B) Difference map between cytosolic A-/B-capsids and nuclear A-capsids, superimposed onto the nuclear A-capsids average. (C, D) Close-up view of a vertex in (A) and (B), respectively. The isosurface thresholds for the difference maps are 1.5σ above the mean density in (A) and 0.5σ in (B).
Figure 5.
Enveloped virions in middle regions of axons.
(A) Schematic diagram of a neuron indicating the mid-axon region. (B) Cryo electron microscopy (projection image) of a pair of enveloped virions (boxed areas) in a mid-axon region vitrified at 16 h p.i.. Note the bundle of microtubules entering and leaving this area. Bar: 200 nm. (C) CryoET slice through the respective reconstructed tomographic volume for the field shown in (B). Asterisk: enveloped capsids; mt: microtubule; pm: plasma membrane. Bar: 200 nm.
Figure 6.
Secondary envelopment at axon terminals.
(A) Diagram of a neuron indicating an axon terminal. (B) Cryo electron microscopy (projection image) of an intact axon terminal at 16 h p.i.. Bar: 200 nm. (C) Slice of the tomogram taken in the area highlighted in (B), showing secondary envelopment of capsids. Asterisks: capsids; white arrow: enveloping vesicle; arrow head: glycoproteins; black arrows: tegument; pm: plasma membrane. Bar: 100 nm. (D) Surface rendering of the tomogram shown in (C). Capsid (light blue), tegument (orange), glycoproteins (yellow), actin (red), plasma membrane and vesicle membrane (blue).