FIG 3.

The SERINC5-mediated induction of proinflammatory cytokines in MDMs is not mediated by the HA tag and independent of viral tropism. (A, upper panel) Relative infectivity of R5-tropic HIV-1 particles produced in 293T cells transfected with a SERINC5 FLAG-tagged expression plasmid or a control vector and analyzed as described in the legend to Fig. 1A. (Lower panel) Representative Western blot analysis of these virions for incorporation of SERINC5.FLAG and HIV-1 p24. (B) Schematic of experimental flow as in Fig. 2B. (C) Relative intracellular levels of p24 as quantified by immunofluorescence (with ΔNef set as 100% and 0.2 to 4% p24⁺ cells). Shown are data points from cells of individual donors, with the mean indicated by a black line. (D and E) Effect of virion incorporation of S5.FLAG on the production of individual cytokines (IL-6 in panel D and IL-8 in panel E) presented as fold change over ΔNef (set to 1). Shown are data points from cells of individual donors, with the mean indicated by a black line. (F) Activated CD4⁺ T cells were infected with the R5 HIV-1 variants from Fig. 2A. Intracellular p24 levels are relative to WT-infected cultures (set to 100%, with the percentage of p24⁺ cells ranging from 16% to 27% between donors). Shown are data points from cells of individual donors, with the mean value of all donors indicated by a black line. (G) Relative levels of intracellular MxA shown as fold change over noninfected (NI; set to 1). (H) MDMs were infected with X4 HIV-1 variants from Fig. 1A. Quantification of IL-6 in the supernatant of MDMs is displayed as relative to ΔNef (set to 1). Shown are data points from cells of individual donors, with the mean of all donors indicated by a black line. (I) Quantification of IL-8 concentration. The fold change between the ΔNef ± S5 conditions is indicated. Statistics (Student's t test): n.s., nonsignificant; **, P < 0.01.