FIG 6.

Sorted viruses are single viral particles. (A) Extracellular virions were purified from cells infected with VP16-GFP or GFP-VP22 viruses, analyzed, and then sorted by flow cytometry as described for Fig. 5A for their low or high levels of VP16 or VP22 content, respectively. All images are only GFP⁺ particles, i.e., those already depleted of aggregates and nonfluorescent components. FSC-H and FSC-A are the height and area under the curve of the forward-scattering signal, respectively, while SSC-A measures the area under the side-scattering signal. Orange, particles with the lowest 10% GFP signal; blue, particles with the top 10% GFP signal; black, particles exhibiting an intermediate GFP signal and not subsequently analyzed. The linear edges around the dot blots reflect the gate used to sort the samples. (B) Extracellular virions purified from infected cells were first diluted 1:50, and then serial dilutions of 1:2 were prepared and analyzed by flow cytometry by continuous recording of events during a fixed time (60 s in these experiments). (C) Sorted viruses were concentrated on 0.1-μm filters, which were embedded and processed for electron microscopy (see Materials and Methods). For clarity, the presence of some capsids with their envelope is indicated by asterisks. Bars represent 100 nm.