Fig. 4.

Differential ISG expression in latently vs productively infected Jurkat cells. a Diagram of fluorophore expression distinguishing Red-Green-HIV (RGH) virus integration vs. replication. mCherry is stably expressed by the CMV promoter, and GFP is conditionally expressed by the HIV LTR promoter upon HIV replication. b Schematic of the RGH virus transduction and sort protocol. Jurkat cells were mock treated, or were transduced with a ΔINT control virus or the RGH virus at MOI 0.2, then after 5d were sorted based on mCherry and GFP fluorophore expression according to the gating scheme shown in c (see also Additional file 1: Figure S2b). Sorted cells were rested in culture for 24 h, then cultured with media alone (untreated control), stimulated with IFNβ, or reactivated wtih PMA/ionomycin, and RNA analyzed by qRT-PCR. d qRT-PCR analysis of HIV RNA in transduced and sorted Jurkat cells treated with media (white bars) or 100 IU/ml IFNβ (blue bars). e qRT-PCR analysis of baseline ISG expression in transduced, sorted Jurkat cells. f qRTPCR analysis of IFN-induced ISG expression in transduced, sorted Jurkat cells treated with 100 IU/ml IFNβ for 8 h. g qRT-PCR analysis of transduced, sorted Jurkat cells treated with media (black symbols) or reactivated with 16 nM PMA and 1 μM ionomycin for 24 h (red symbols). For all qRT-PCR analyses, fold change (FC) was calculated relative to untreated, mock-transduced cells (ΔΔCt method), and each symbol represents FC of an individual biological replicate cultured and treated separately within single experiment (three replicates per treatment condition). Bars represent mean mean FC + SD. Statistical significance relative to mock control cells was calculated by twoway ANOVA (Holm-Sidak); asterisks denote significance (*p < 0.05, **p < 0.01, ***p < 0.001)