The SFN-mediated HIV infection block is reversible.

<p>(A), Study design: PMA-differentiated THP1 cells were pretreated with media supplemented with vehicle only (DMSO) or with 10 μM, 20 μM or 30 μM SFN. After twenty-four hours of treatment, the culture media was replaced with fresh SFN-free media. Cultures were infected 1, 2, 3, 4, or 5 days after treatment with freshly thawed aliquots of VSV-G pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i>. Twenty-four hours after the start of each time-point infection, the cells were harvested and (B), luciferase activity was measured by photon emission. The bar graph represents the data for replicate experiments (n = 3).</p

SFN blocks infection after entry and but before 2-LTR circle formation.

<p>Replicate cultures of hMDMs were pretreated with vehicle (DMSO)-containing media, with 5 μM AZT or with 10 μM SFN. Twenty four hours after treatment, the samples were infected with VSV-G-pseudotyped HIV-1 encoding GFP in place of <i>nef</i>. Cultures treated with heat-inactivated virus served as controls for plasmid carry over and for impaired viral entry. Cells were harvested and DNA was isolated 24 hours after infection. Viral DNA products were detected by real-time PCR using primer sets specific for the indicated stage of reverse transcription. (A), Relative quantities of late reverse transcription products, (B), 2-LTR circles, and (C), integrated proviruses. The bar graph represents the data for replicate experiments (n = 3).</p

SFN action impacts HIV-2 as well as HIV-1 and is not reporter dependent.

<p>PMA-differentiated THP1 cells were treated with media supplemented with vehicle only (DMSO), 5 μM AZT or with 10 μM SFN. Twenty-four hours after treatment, the samples were either mock infected or infected with (A), VSV-G pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i> or (B), VSV-G pseudotyped HIV-2 encoding firefly luciferase in place of <i>nef</i>. Twenty-four hours after infection, luciferase activity was measured by photon emission. (C), In parallel, the same experiment as in (A) and (B) was performed except that THP1 cells were infected with VSV-G-pseudotyped HIV-1 with GFP in place of <i>nef</i> or (D), VSV-G pseudotyped HIV-2 with GFP in place of <i>nef</i>. The samples with GFP-reporter viruses were fixed and harvested 24 h after infection and the fraction of GFP(+) cells was enumerated by flow cytometry. Bar graphs represent the data for replicate experiments (n = 3).</p

SFN action blocks spreading infections that rely on the HIV envelope for viral entry.

<p>hMDMs were pretreated with vehicle (DMSO), 5 μM AZT, or 10 μM SFN. All cultures were subsequently maintained in their respective treatments for the duration of the experiment. Twenty four hours after initial treatment, the cultures were infected with the HIV-1 clinical isolate 89.6. Culture supernatants were collected 3, 6, 9, and 14 days after infection. (A), Western blots and (B), p24 antigen ELISA assays of viral supernatants were performed. (C), Fourteen days after infection, cells were imaged using phase contrast microscopy. (D), Uninfected replicate cultures were maintained in the presence of vehicle (DMSO) or in 10 μM SFN. After 14 days of treatment, the viability of each cell type was assessed under each condition by measuring water-soluble tetrazolium salt (WST-8) formazan reagent cleavage by cellular dehydrogenases. Continuous treatment of cells with 10 μg/ml of the eukaryotic toxin blasticidin served as a positive control to demonstrate loss of viability. (E), hMDMs were infected with 89.6-Env-pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i>. The bar graphs represent the data for replicate experiments (n = 3).</p

SFN blocks HIV infection in monocytoid cell lines but does not increase SAMHD1 expression.

<p>(A), HeLa, (B), GHOST, (C), HEK293T, (D), PMA-differentiated U937, and (E), PMA-differentiated THP1, cells were treated with media supplemented with vehicle only (DMSO) or with 10 μM, 20 μM or 30 μM SFN. Pretreatment of cell cultures with 5 μM AZT served as a positive control for viral inhibition. Twenty-four hours after treatment, the samples were infected with VSV-G pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i>. Twenty-four hours after infection, the cultures were harvested and luciferase activity was measured by photon emission. (F), PMA-differentiated THP1 cells were treated with SFN that underwent a twofold serial dilution with 10μM of SFN being the highest concentration. Twenty-four hours after treatment, the samples were either mock infected or infected with VSV-G pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i>. Twenty-four hours after infection, luciferase activity was measured. The bar graphs represent the data for replicate experiments (n = 3). (G), SAMHD1 protein was detected by western blot analysis from lysates of mock- and SFN treated PMA-differentiated U937 cells. Lysates from mock-treated, PMA-differentiated THP1 cells served as a positive control for SAMHD1 production.</p

SFN does not trigger expression of interferon-stimulated anti-viral factors SAMHD1 or MX2.

<p>PMA-differentiated THP1 cells were mock-treated or treated with media supplemented with vehicle only (DMSO) or with 10 μM SFN or with 500 U/mL of IFNα. (A), Proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. (B), Densitometric analysis was performed on the Nrf2, SAMHD1, MX2, NQO1 and GCLM (NQO1 and GCLM are both indicators of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the bands were then graphed.</p