1. Resistance of CD141+ DCs to HIV and influenza virus infection at the level of viral fusion.
Resistance of CD141+ DCs to HIV and influenza virus infection at the level of viral fusion. (A) HIV-1 fusion assay in blood DCs. Viral fusion revealed by CCF4 fluorescence in blood DCs after infection with HIV-1(BaL) (MOI = 0.8) or HIV-1(NL4-3) (MOI = 0.6) containing a BlaM-Vpr fusion protein. Fluorescence of the CCF4 product indicates viral fusion with target cells as a result of cleavage of the cell-loaded CCF4 substrate by the virus-contained BlaM. (B) Quantification as in (A) (n = 8 donors combined from four independent experiments). (C) Staining of GFP proteins contained in viral particles and CD44 in CD1c+ and CD141+ DCs after infection with HIV-1(V3R5) iGFP containing GAG-iGFP and GFP-Vpr fusion proteins, alone or in the presence of viral entry inhibitors MVC and TAK-779. Scale bars, 10 μm. (D) Quantification of the GFP density in GFPlow regions as in (C), shown for one representative donor (top) and average for five donors (bottom; combined from two independent experiments). (E) Levels of influenza virus receptors on blood DCs. SNA and MAA binding on CD1c+ and CD141+ DCs (representative of two independent experiments). (F) GFP expression in blood DC subsets that were sorted and infected for 48 hours with GFP-coding lenti(H1N1) Vpx at MOIGHOST X4R5 = 1 (n = 4 independent donors combined from two independent experiments). Viruses were not spinoculated. (G) Viral fusion revealed as in (A) by CCF4 fluorescence in blood DCs after infection with lenti(H1N1) (MOI = 1) containing a BlaM-Vpr fusion protein (n = 4 donors combined from two independent experiments). Viruses were not spinoculated. (H) GFP expression in blood DC subsets that were sorted and infected for 24 hours with HSV-1–GFP at MOI = 25 (one representative donor). (I) Quantification of GFP expression and frequency of live cells in blood DC subsets that were sorted and infected for 24 hours with HSV-1–GFP at MOI = 25 as in (H) (n = 10 combined from four experiments). (J) GFP expression in blood DC subsets that were sorted and infected for 24 hours with enhanced GFP–expressing VSV (VSVeGFP) at MOI = 16 (one representative donor). (K) Quantification of GFP expression and frequency of live cells in blood DC subsets that were sorted and infected for 24 hours with VSVeGFP at MOI = 16 as in (J) (n = 5 combined from two experiments). (L) Viral fusion revealed as in (A) by CCF4 fluorescence in blood DCs after infection with lenti(G) (MOI = 10) containing a BlaM-Vpr fusion protein (n = 13 combined from five experiments). ns, not significant; SSC, side scatter. *P < 0.05, **P < 0.01, ***P < 0.001, ****P <
Aymeric Silvin et al. Sci. Immunol. 2017;2:eaai8071
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