Figure 7. Effect of Sp1 inhibition and PYHIN proteins on retroviral production in vitro and in vivo.

(A) J-Lat cells (clone 10.6) were stimulated with the indicated doses of TNFα in the presence of increasing doses of Mithramycin A. Two days post-stimulation, GFP expression and MFI were determined by flow cytometry. The middle and right panels show mean values (±SEM) derived from three independent experiments.

(B) GFP expression in different clones of J-Lat 10.6 cells lacking IFI16 or Sp1 expression was determined two days post-stimulation with TNFα (1 ng/ml) and Mithramycin A (20 nM) by flow cytometry. Mean GFP fluorescence intensities (±SEM) derived from three independent experiments are shown. Asterisks indicate differences compared to the control cells (n.t.). * p < 0.05, ** p < 0.01, *** p < 0.001.

(C) Luciferase activities in PMA-stimulated or unstimulated CD4+ T cells from two donors latently infected with VSV-G pseudotyped HIV-1 nano-luc reporter constructs in the absence or presence of Mithramycin A.

(D) HEK293T cells were cotransfected with a retrotransposition-competent (L1) or -defective (L1 neg) LINE-1-GFP reporter plasmid and a vector control or expression constructs for IFI16 or SAMHD1. Five days post-transfection, GFP+ cells were quantified by flow cytometry (n=3 ± SEM).

(E) HEK293T cells were cotransfected with LINE-1 or CMV promoter constructs driving luciferase gene expression and expression constructs for IFI16 or a control vector. Luciferase activities (relative light units, RLU) in cell lysates were determined two days later (n=3 ± SEM).

(F) Effects of human IFI16 or AIM2 and murine p204 on (left) infectious virus yield and (right) LTR-driven eGFP expression of HIV-1 in HEK293T cells 40 hours post-transfection. Values represent mean levels of infectious virus production or eGFP expression (±SEM; n=3) in the presence of increasing doses of the indicated PYHIN proteins relative to the vector control (100%).

(G) Expression of HA-tagged PYHIN proteins and co-expressed BFP in cells from (F) was analyzed by Western blot.

(H) HEK293T cells were cotransfected with increasing doses of expression constructs for p204 and a proviral MLV construct. At 40 h post-transfection, MLV RT activity in cell culture supernatants was determined. Shown are mean values (±SEM) derived from three independent experiments.

(I, J) Viral loads in wild type and ALR−/− mice infected with Friend virus. Wild-type C57BL/6 and ALR−/− mice lacking all 13 genes encoding AIM2-like receptors (also name PYHIN proteins; Gray et al., 2016) were infected with 20,000 SFFU (spleen focus-forming units) of Friend virus. At 3 dpi, (D) plasma viremia was determined and (E) spleens were harvested to analyze viral loads in an infectious center (IC) assay. Thirteen mice per group from at least two independent experiments were analyzed. Mean (±SEM) values are indicated by bars. Statistically significant differences between the groups were analyzed using a Mann-Whitney test and are indicated by * for p < 0.05 and ** for p < 0.01.