Extended Data Figure 8: Inhibition of HIV-1 with cyclosporine or by TREX depletion triggers IFN production. | Nature

Extended Data Figure 8: Inhibition of HIV-1 with cyclosporine or by TREX depletion triggers IFN production.

From: HIV-1 evades innate immune recognition through specific cofactor recruitment

Extended Data Figure 8

a, Infection of human MDM with WT NL4.3 (Ba-L Env) in the presence or absence of 5 μM cyclosporine. Cells were stained for p24 at specific time points after infection and infected colonies counted. b, Supernatants isolated from MDMs in a were assayed for soluble IFN-β levels by ELISA. c, MDM were infected at low multiplicity with WT HIV in the presence of cyclosporine and IFNAR2 antibody or isotype cAb. d, Infectious titre of WT HIV was determined on MDM in the presence of DMSO or cyclosporine at 48h post infection. The data are presented as infectious units (iu) per nanogram (ng) of reverse transcriptase (RT) measured by ELISA. e, To confirm that SmBz-CsA inhibits recruitment of cyclophilin A (CypA), but not Nup358 Cyp, to HIV-1 CA, we used the TRIMCyp restriction assay. HIV-1 GFP vector titer on CRFK cells expressing empty vector (EV), HA-tagged owl monkey TRIMCyp RBCC domain fused to human CypA (TRIMCypA) or human Nup358Cyp (TRIMNup358) in either DMSO, or 5 µM cyclosporine or 10 µM SmBz-CsA. Protein levels were measured by immunoblot detecting the HA tag with β-Actin as a loading control. In this assay both cyclosporine and SmBz-CsA inhibited CypA recruitment to CA and rescued VSV-G pseudotyped HIV-1 GFP infectivity from restriction by TRIMCypA. However, neither drug rescued HIV-1 infectivity from TRIMNup358, confirming cyclosporine specificity for CypA and not Nup358. f, TREX-1 expression was determined in MDM expressing TREX-1 specific shRNA or control shRNA by qRTPCR, normalized to GAPDH at the time of HIV-1 infection in g. g, Cells were stained for p24 at specific time points after WT HIV-1 infection of TREX-1 depleted and control shRNA expressing MDM and infected colonies counted. h, Supernatants isolated from MDM in g were assayed for soluble IFN-β by ELISA. i, Infection of TREX-1 depleted MDM with WT HIV-1 at low multiplicity in the presence of either IFNAR2 antibody or isotype cAb. Cells were stained for p24 at specific time points after infection and infected colonies counted. j, Hairpin transduced MDM were assayed for the production of soluble IFN-β levels before HIV-1 infection. Data are representative of 3 independent biological replicates. For HIV-1 replication assays in MDM data points and nonlinear regression lines over time are shown.

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