FIG 4.

Tegument-capsid susceptibility to detergent extraction. (A) Dual-fluorescent extracellular viral particles containing the pUL25/mCherry capsid tag and GFP fused to a designated tegument protein were purified from the supernatant of infected cells and treated with buffer lacking (−) or containing (+) Triton X-100. Representative images of capsid and tegument emissions are shown, with each channel scaled equally across all samples. (B) GFP emission intensities from individual particles are show as histograms. The percentages in the upper right corner of each histogram indicate the proportion of particles with detectable GFP signal above threshold following detergent extraction. The data were fit to either Gaussian or decaying exponential distributions. All distributions had goodness-of-fit (R²) values of >0.95. (C) The red and green fluorescent signals of tandem-tagged mRFP-GFP-pUL35 particles were measured both before and after Triton X-100 extraction, and a with/without Triton X-100 detergent ratio was calculated for each fluorescence channel (n = 3). (D) Average red capsid (mCherry/pUL25) fluorescence intensities from purified particles following mock or Triton X-100 extraction (n = 3 per condition). Error bars indicate standard errors of the means. The solid horizontal line is the average intensity (intact plus mock extracted) across the seven capsid-tegument dual-fluorescent viruses. The dotted horizontal line is the average intensity following detergent extraction. (E) Estimated protein copy numbers based on average green fluorescence intensities (see Materials and Methods). Error bars indicate standard errors of the means.