Representative virus-like arrays of 25-nm particles in N2a + 22L (A–C) and in GT+FU-CJD (D) cells. (A and B) Particles in healthy cells treated with 100 ng/ml PMA for 5 days when PrP-res was markedly increased, with higher magnifications of the same array (Right). (C) From an N2a + 22L culture without PMA, processed in parallel. (D) A comparable virus-like array in a GT+FU-CJD cell. (A Left) The membrane-bound array is distant from the nucleus (N) and sits between a mitochondrion (m) and a vacuole (V). A 100-nm-diameter IAP retroviral is seen in an RER cistern (arrow), and Golgi membranes are at g. Arrows in the corresponding magnified image point to spherical dense particles of ≈25 nm; note regions of variable compaction of particles. (B) A characteristic cluster of IAP retroviruses is seen to the right of a vacuole (V) in addition to a TSE-packed particle array adjacent to an isolated IAP (arrow). Higher magnification shows that the paracrystalline particles have sharper outlines than adjacent ribosomes. (C) Without PMA, spherical 25-nm particles also line up in an orthogonal pattern in more compact regions of N2a + 22L cells. (D) Orthogonal or paracrystalline arrangement of equivalent dense 25-nm particles in cross-section (e.g., at arrows) in infected GT cells that produce no retroviral structures is shown; a few similar 25-nm particles are seen in an adjacent swollen RER cistern (arrowhead), and these may represent a different stage of array development (see Results). (Scale bars: 100 nm.)

Relationship of PrP-res, virus-like particles, and infectivity in N2a + 22L cells grown with and without PMA. (A) Dose–response from 0 to 100 ng/ml PMA for 5 days of PrP and corresponding PrP-res amounts (+Pk lanes of whole-cell lysates treated with proteinase K as described in ref. 4). Note the strong increase in PrP-res at 100 ng/ml. CNS is brain homogenate control. MK are mock-infected cells and show no PrP-res. (B) PrP-res during continuous treatment with 100 ng/ml for 1–7 days shows the strongest response at 5 days; cells were treated in parallel and collected at sequential times. (C) Quantitative analysis of the increase in PrP-res in seven independent experiments with 75–100 ng/ml PMA (dotted line). Note the reproducible rise at 5 days (± SEM). The gray line shows the PMA-induced PrP-res increase versus a parallel nondrug control (see E) in cells that were tested for infectivity. (D) Count of virus-like 25-nm particle arrays in PMA-treated mock (uninfected N2a) control cells and in N2a + 22L cells without (22L-PMA) and with (22L+PMA) the drug. More than 250 cells were counted by EM for each sample, and the inclusion of any questionably positive arrays makes the percentage of positives overestimated. Nevertheless, there is no significant increase in the number of particle arrays with PMA. (E) Western blot of an aliquot of the mock-infected, untreated 22L, and PMA-treated 22L cells inoculated in mice for infectivity titrations. The mock cells show no PrP-res (+PK lanes), and the PMA-treated cells show an obvious increase in both PrP and PrP-res compared with their untreated counterparts. All cell lanes loaded with equal amounts of protein and infected CNS show brain-specific pattern (4). (F) Incubation time after inoculation of serial dilutions of N2a + 22L cell homogenates grown without PMA (white bars) and with PMA (gray bars). A shorter incubation time indicates a higher infectious titer. Mock-infected cells (dotted bar) at high concentrations elicited no clinical signs or neuropathology.

Ultrasmall immunogold labeling (silver intensified) of PrP and PrP-res in N2a + 22L cells lightly fixed and then permeabilized in situ for antigen detection. (Top) A typical low-power image where many grains are on the plasma membrane (arrowhead) and dispersed in the cytoplasm. Nuclei (N) and clusters of IAP retroviruses (arrow) are not labeled. (Middle) Grains over PrP amyloid fibers in characteristic crisscrossing bundles (A) and also over RER membranes (arrows). Label was not seen over mitochondrial cristae (m) as representatively depicted. (Bottom) PrP does not localize over the 25-nm virus-like dense particles but can be associated with the outer RER membrane. (Scale bars: 100 nm.)