Figure. 4. Enrichment of HIV pseudoviruses with PE overcomes restriction of fusion of Serinc5-containing particles by decreasing membrane heterogeneity and order.

HIV pseudoviruses with or without Serincs were enriched with the lipids listed on the X-axis, separated from free lipids, and (A) fusion with supported plasma membranes was assessed by TIRF microscopy-based single-particle membrane fusion. A two-way ANOVA test was conducted to examine the statistical significance of the effects of Serinc incorporation and lipid enrichment on HIV pseudovirus fusion. There was a statistically significant interaction between the two factors, F(14, 88)=4.894, p<0.001. Tukey’s multiple comparisons test showed very significant differences in the means of the comparisons shown above (**** p<0.0001, ns not significant). None of the No Serinc-Serinc2 comparisons were significant. Each data point represents the fraction of particles fused on a separately prepared supported membrane. Each condition includes data from at least three distinct preparations of pseudovirus. (B), Fluorescence lifetimes of FLIPPER-TR in lipid-enriched, Serinc5-containing HIV pseudoviruses. HIV pseudoviruses containing Serinc5 were stained with FLIPPER-TR after enrichment with the lipid listed on the X-axis and imaged by fluorescence microscopy as in Figure 1. Each data point is the result obtained from one field of view within a sample and is plotted with mean and standard deviation. Statistical significance by Welch’s unpaired t-test: * p<0.05, ns not significant. (C) Normalized distributions of membrane thickness (D_TT) measurements of 5 nm segments of the same preparation of Serinc5-containing HIV pseudoviruses enriched with the lipid listed in the key as panel (B) and their corresponding double-Gaussian fits. The unenriched Serinc5 distribution is replotted from Figure 2E for comparison.