FIG 5.

In situ hybridization, after long incubation times, of BSC-1 cells infected at a low MOI. (A) Cells were infected at an MOI of 1 for 10 min with doubly labeled rcTLPs and washed, and infection was allowed to continue for 5 h. Paraformaldehyde fixation was followed by overnight incubation with the pool of 44 Atto 565-labeled oligonucleotide probes and subsequent 3D imaging at 640-nm (left), 488-nm (middle), and 561-nm (right) excitation wavelengths, maximum-intensity projections of which are shown. (B) The uncoated DLP highlighted in the red box in panel A, displaying the individual channels in the xz and yz planes through the particle, as well as the maximum projection of the overlay of all three channels. (C) The uncoated DLP highlighted in the green box in panel A (white arrow), displaying the overlay of all three channels and the individual channels in the xz and yz planes through the center of the particle. For comparison, the yz plane of a nearby particle that has retained its VP7 shell (yellow arrow) is also shown. (D) Scatter plot of the number of Atto 565 probes colocalized with a given uncoated DLP after 5 h of infection at an MOI of 1 (○) or 5 (△). The uncoated DLPs highlighted in panels B and C are represented here as red and green circles, respectively, plotted within the data collected from cells infected at an MOI of 1. Quantification of the number of colocalized Atto 565-labeled probes was performed as described in Materials and Methods.