Figure 2.

IEVs separated from free virions are able to infect cells. (a and b) The iodixanol gradient purification of IEVs was performed for samples produced at different time points after the initial infection of the cells. Cytotoxicity of the 10 fractions was assessed using the sample originating cell lines as the recipients. In addition to the free virus containing fractions, also lighter fractions 2–6, corresponding to the fractions where IEVs, were found cytotoxic. (c–e) In order to clarify, whether IEVs could induce the formation of new viruses in recipient cells, the cells were incubated with an autologous IEV fraction 4 of D1 and D3 and imaged using TEM. In both cell lines (c: A549 after 48 h with IEV fraction D3, d: PC-3 after 24 h with IEV fraction D3 and e: A549 after 48 h with IEV fraction D1), viruses (red arrows) were found in the nuclei of the treated cells. The nuclear membrane is shown in the images with a white arrowhead. In a and b, the median viability compared to the untreated control cells is presented with individual results, with sample size N = 3. Statistical significance was assessed with one-way ANOVA with Tukey’s HSD post hoc test in a and b, and denoted by grouping with letters: for all variables with the same letter, the difference between the means is not statistically significant. If two variables have different letters, they are significantly different. Uppercase letters denote fractions of the same time point while lower case letters denote different time points of each fraction. Additionally, statistically significant difference to the non-treated control samples is denoted with an asterisk. p-values of statistically differing samples are listed in Supplementary Figure 5.