Membrane-Associated Enteroviruses Undergo Intercellular Transmission as Pools of Sibling Viral Genomes

Figures

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  Figure 1. Fast-Sedimenting CIUs Contain Membrane-Associated Virion Pools (A) Scheme of the process used to separate CIUs from free virions by low-speed centrifugation. Three serial centrifugation and resuspension steps were carried out in which we separated the supernatant (S) and pellet (P) fractions. P fractions were subjected to Triton detergent to disrupt membranes and release free virions (B fraction). (B) Transmission electron micrographs of membrane-associated virions obtained from the P3 fraction. The small white bars correspond to 30 nm, the diameter of enterovirus virions. The observed sizes of virion-like structures are close to this size, although often smaller, which is expected if the virion section is not diametrical. (C–E) Infectivity of the indicated fraction quantified by the plaque assay. The mean and SEM (error bars) titers obtained from three independent assays are shown. (C) Culture media harvested at 12 hpi. (D) Culture media harvested at 8 hpi. (E) Culture media harvested at 12 hpi in which the P2 fraction was purified using annexin V (P2∗ and B2∗).

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  Figure 2. Infrequent Co-transmission of EGFP- and mCherry-Encoding CVB3 Viruses through CIUs (A) Flow cytometry analysis of producer cells inoculated at high density (D ≈ 10 PFUs/cell) with a 1:1 mix of the two variants (culture media harvested at 12 hpi). (B) Flow cytometry analysis of cells inoculated at lower density (D ≈ 0.1 PFU/cell) with the P3 fraction. Results for one replicate are shown. Cell counts for three replicates are shown in Table S1. (C) Distribution of the estimated proportion of each variant in individual producer cells (g), rescaling the intensities shown in (A) such that the average rescaled intensities matched the observed proportion of cells infected with each variant (46.4% ± 0.4% CVB3-EGFP versus 53.6% ± 0.4% CVB3-mCherry; Table S1). This was achieved by multiplying all EGFP intensities by 1.82. The resulting coefficients of variation of mCherry and rescaled EGFP intensities were similar (0.72 and 0.68, respectively). (D) Boxplot of the probability that CIUs containing n infectious particles co-transmitted both variants, assuming random mixing. This probability was calculated as p(n) = 1 − gn − (1 − g)n. The lower and upper limits of the box indicate 25th and 75th…

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  Figure 3. Co-transmission of CVB3-EGFP and CVB3-mCherry Viruses Determined by Foci Analysis Left: virus-producer cells inoculated at high density (10 PFUs/cell) with a 1:1 mix of each variant, shown at 7 hpi. Green and red channels are shown separately, as well as the merged image. Image saturation was maximized so as to reveal the presence or absence of each variant, rather than quantitative information of infection level. Scale bars, 0.3 mm. Right: analysis of infection foci produced in recipient cells inoculated at limiting dilution. A 96-well plate inoculated with the P3 fraction and imaged by whole-well automated microscopy at 30 hpi is shown. Green, red, and yellow regions correspond to EGFP, mCherry, and doubly fluorescent cells, respectively. A magnified view of two wells at an earlier time point is also shown (16 hpi). Top: two independent foci in the same well. Bottom: two variants initially co-transmitted as part of the same infectious unit. This co-transmission was short-lived, as shown by the fact that most cells in the resulting foci were infected with only one variant. Scale bars, 1 mm.

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  Figure 4. Mutation Frequency across the CVB3 Genome The mutation frequency (mutated/total bases read) per nucleotide site is shown for the founder and one evolution replicate (line 1) for each treatment (S1, P3, and B3). Replicate evolution lines 1 and 2 showed very similar genome-wide diversity patterns (data available from Table S4).