Increased iron export by ferroportin induces restriction of HIV-1 infection in sickle cell disease by Namita Kumari, Tatiana Ammosova, Sharmin Diaz, Xionghao Lin, Xiaomei Niu, Andrey Ivanov, Marina Jerebtsova, Subhash Dhawan, Patricia Oneal, and Sergei Nekhai BloodAdv Volume 1(3):170-183 December 27, 2016 ©2016 by American Society of Hematology

Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology

Inhibition of HIV-1 in SCD-derived PBMCs Inhibition of HIV-1 in SCD-derived PBMCs. (A) HPLC analysis of SCD hemoglobin. Inhibition of HIV-1 in SCD-derived PBMCs. (A) HPLC analysis of SCD hemoglobin. HPLC of representative blood samples obtained from an SCD subject with HbSS hemoglobin and normal control with HbAA hemoglobin. (B-C) Inhibition of 1 round of HIV-1 infection in SCD-derived PBMCs. PBMCs were purified from whole blood obtained from SCD patients and healthy controls. The cells were activated with PHA and IL-2 and infected with HIV-1-LUC-G virus expressing luciferase. Luciferase activity was measured at 24 hours postinfection (PI) and normalized to the cell numbers. (B) A representative sample that corresponds to panel A. (C) Comparison of 1 round of HIV-1 replication in a cohort of 15 SCD patients. The results are expressed relative to infection of PBMCs from 9 control subjects, which were set to 100%. The means ± standard error (SE) and P value calculated with Student t test are shown. (D) Inhibition of p24 production in SCD-derived PBMCs. PBMCs obtained from 3 SCD patients and 3 controls were activated and infected as described above. Supernatants were collected 72 hours PI, and p24 was measured by ELISA. The means ± SE and P value calculated with Student t test are shown. (E) Inhibition of HIV-1 env expression in SCD-derived PBMCs. PBMCs obtained from 4 SCD patients and 4 controls were activated and infected as described above for 48 hours. RNA was extracted, reverse transcribed, and analyzed with primers for HIV-1 env gene by RT-PCR using 18S RNA as a reference. The means ± SE and P value calculated with Student t test are shown. (F) Inhibition HIV-1 RT in SCD PBMCs. PBMCs obtained from 3 SCD patients and 3 controls were activated with PHA and IL-2 and infected with HIV-1-LUC-G. At 6 hours PI, DNA was extracted and analyzed by RT-PCR on Roche 480 using primers for early LTR and β-globin gene as a reference. The means ± SE are shown (n = 3 for each sample). The means ± SE and P value calculated with Student t test are shown. Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology

Altered iron metabolism in SCD-derived PBMCs is critical for HIV-1 inhibition. Altered iron metabolism in SCD-derived PBMCs is critical for HIV-1 inhibition. (A) Heat map showing expression of selected iron and HIV-1 regulatory genes in SCD PBMCs. RNA was extracted from PBMCs obtained from 3 SCD patients and 3 controls. RNA was reverse transcribed and analyzed by RT-PCR using primers for BACH1, CDK2, Cyclin A, Cyclin E, EGR1, ferroportin, hepcidin, HIF-1α, HO-1, IKBα, p21, p27, PP1α, and SAMHD1. The 18S RNA was used as a housekeeping control gene. Heat map was constructed using Morpheus software. P values were determined using paired Student t test. (B) Validation of candidate genes expression. Expression of IKBα, HO-1, p21, HIF-1α, and ferroportin mRNA was analyzed from additional 3 controls and 7 SCD PBMCs. SAMHD1 expression was analyzed in 3 controls and 6 SCD PBMCs. RNA was isolated, reverse transcribed, and analyzed by real time. 18S rRNA was used as a reference for ΔΔCt analysis. The means ± SE and P value calculated with Student t test are shown (C) IPA of the iron and HIV-1 regulatory genes. Ingenuity software analysis of genes shown in panel A identified a protein network that connected iron and HIV-1 regulatory genes through virus replication, anemia, and cell cycle progression networks (D). Upregulated genes are colored in red and downregulated genes are colored in green. (D) IPA of viral and pathogen restricting genes in SCD. Ingenuity software analysis of 250 upregulated and downregulated genes determined by meta-analysis of Geoset GSE53441 (data from PBMCs of 24 SCD patients and 10 controls) identified a protein network of 12 genes involved in antiviral response and viral replication. (E) Hepcidin restores HIV-1 replication in SCD PBMCs. Activated PBMCs from 3 SCD patients and 3 control individuals were infected with HIV-1-LUC-G virus and treated with 0.9 μM hepcidin, 20 μM ferric ammonium citrate (iron), or 10 μM PPYeT iron chelator. At 48 hours PI, luciferase activity was measured. The means and P values determined by Student t test are shown. (F) Hepcidin in plasma from SCD patients. Hepcidin was measured in plasma obtained from 13 SCD and 12 healthy subjects by HR/SIM method. The means and P values determined by Student t test are shown. Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology

Deregulation of intracellular iron in SCD PBMCs Deregulation of intracellular iron in SCD PBMCs. (A) Increased ferritin expression in SCD PBMCs. PBMCs obtained from 3 SCD patients and 3 controls were cultured in media supplemented with PHA for 36 hours and then activated with IL-2 for 36 hours. Deregulation of intracellular iron in SCD PBMCs. (A) Increased ferritin expression in SCD PBMCs. PBMCs obtained from 3 SCD patients and 3 controls were cultured in media supplemented with PHA for 36 hours and then activated with IL-2 for 36 hours. Where indicated, control PBMCs were further treated with 100 μM hemin for 24 hours. Ferritin was measured in cell lysates by ELISA. The means ± SD are shown. (B) Reduced intracellular iron levels in SCD PBMCs. Labile intracellular iron pool was measured in PBMCs obtained from 3 SCD patients or 3 normal controls. Cells were treated with 0.1 μM calcein AM for 10 minutes at 37°C. After washing with PBS, cells were incubated at 37°C, and calcein fluorescence was measured in a Glomax Multidetection system at different time points. Fractional fluorescence (F − F0)/F, which is inversely proportional to the amount of chelatable iron plotted on the y-axis. Paired Student t test was used to determine P value of the iron values at 30 minutes. (C-G) Elevated expression of TFR, HIF-1α, ferroportin, IKBα, and HO-1 in SCD PBMCs. Cell lysates from activated PBMCs obtained from SCD patients and normal controls were resolved on 4% to 12% Bis-Tris gel and probed with antibodies against TFR, HIF-1α, ferroportin (FPN), IKBα, and HO-1. The β-actin was used as loading control. Results were quantified using Image Quant Software. Bars represent independent experiments on 4 patients and 4 controls for HIF-1α and IKBα; 6 SCD patients and 6 controls for ferroportin; and 3 SCD patients and 3 controls for HO-1. The blots show 2 representative SCD and control samples. The means ± SD are shown. P values were calculated using Student t test. Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology

FPN, hepcidin, and iron regulatory genes play a role in heme-mediated HIV-1 inhibition. FPN, hepcidin, and iron regulatory genes play a role in heme-mediated HIV-1 inhibition. (A) Hemin treatment induced ferroportin expression. THP-1 cells were treated with hemin for 24 hours. Cell lysates were resolved on 4% to 12% Bis-Tris gel and probed with antibodies against FPN. The β-actin was used as loading control. Results were quantified using Image Quant Software. Bars represent quantification from 4 SCD and control samples. The means ± SD are shown. P values were calculated using Student t test. (B) Expression of candidate genes in hemin-treated THP-1 cells. THP-1 cells were treated with hemin for 24 hours. RNA was extracted, reverse transcribed, and analyzed by RT-PCR using primers for IKBα, HO-1, p21, HIF-1α, and FPN. The18S rRNA was used as a reference for ΔΔCt analysis. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (C) Restoration of hemin-inhibited HIV-1 replication in THP-1 cells and PBMCs. THP-1 cells and PBMCs were treated with hemin or with hemin and hepcidin and infected with HIV-1-LUC-G. Luciferase activity was measured 2 days PI. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (D) Restoration of hemin-inhibited HIV-1 replication in MDMs. MDMs were treated with hemin or with hemin and hepcidin and infected with M-tropic HIV-1 (BAL) isolate. Expression of gag was measured by RT-PCR after 6 days PI. The means ± SD are shown (n = 3 for each sample). P values were calculated using Student t test. (E-I) Restoration of hemin-mediated HIV-1 replication by knockdowns of FPN, IKBα, HO-1, p21, and HIF-1α. THP-1 cells were stably transduced with lentiviruses expressing corresponding shRNA and infected HIV-1-LUC-G virus. Where indicated, the cells were also treated with 75 μM hemin. Luciferase activity was measured 2 days PI (left panels). The expression of the corresponding mRNAs was measured by RT-PCR with 18S RNA primers for internal control (right panels). Quantification from 3 independent experiments show the means ± SD and P values calculated using Student t test. Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology

Reduction of CDK2 activity in SCD PBMCs Reduction of CDK2 activity in SCD PBMCs. (A) CDK2 activity is reduced in SCD PBMCs. PBMCs were obtained from 3 SCD patients and 3 control subjects and activated with PHA and IL-2. Reduction of CDK2 activity in SCD PBMCs. (A) CDK2 activity is reduced in SCD PBMCs. PBMCs were obtained from 3 SCD patients and 3 control subjects and activated with PHA and IL-2. Cells were lysed, and CDK2 was immunoprecipitated using anti-CDK2 antibodies from lysates that were equalized by protein. Kinase assays were performed using histone H1 as a substrate. Upper panels show a representative immunoblot of CDK2, a radioactive image of phosphorylated histone H1, and a Coomassie-stained image of histone H1. Lower panel shows quantification from 3 independent experiments. The means ± SD and P values calculated using Student t test are shown. (B) CDK2 knockdown inhibits HIV-1 infection in THP-1 cells. THP-1 cells were stably transduced with lentiviruses expressing CDK2-targeting shRNA or control shRNA and then infected with HIV-1-LUC-G virus. Left panel shows luciferase activity measured at 48 hours PI. Right panel shows expression of CDK2 mRNA determined by RT-PCR with 18S RNA as an internal reference for ΔΔCt analysis. The means ± SD are shown for 3 CDK2 KD and 3 control shRNA clones. (C) CDK2 activity is reduced in hemin-treated THP-1 cells. THP-1 cells treated with 75 μM hemin and control cells were lysed, and CDK2 was immunoprecipitated using anti-CDK2 antibodies. Kinase assays were performed using histone H1 as a substrate. Lower panel shows quantification from 3 independent experiments. P value was calculated using Student t test. (D) Hemin treatment reduces CDK2 mRNA expression. THP-1 cells were treated with 75 μM hemin for 24 hours. RNA was extracted, reverse transcribed, and analyzed by RT-PCR using primers for CDK2 and 18S rRNA as an internal reference for ΔΔCt analysis. The means ± SD and P values calculated using Student t test are shown for 3 independent samples. N/s abs, nonspecific antibodies. Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology

Reduction of SAMHD1 phosphorylation in SCD PBMCs and hemin-treated THP-1 cells. Reduction of SAMHD1 phosphorylation in SCD PBMCs and hemin-treated THP-1 cells. (A) Reduced SAMHD1 Thr-529 phosphorylation in SCD PBMCs. PBMCs obtained from 4 SCD patients and 4 controls were activated with PHA and IL-2 and treated with 75 μM hemin for 24 hours. Cell lysates were resolved on 4% to 12% Bis-Tris gel and probed with antibodies against SAMHD1 and Thr-592 phosphorylated SAMHD1 (SAMHD1-(P)) and β-actin as loading control. Lower panels show quantification for 4 individual samples. Shown are SAMHD1 normalized to β-actin (upper panel), SAMHD1-(P) normalized to β-actin (middle panel), and SAMHD1-(P) normalized to SAMHD1 expression (bottom panel). The means ± SD and P values calculated using Student t test are shown. (B) Increased SAMHD1 expression and reduced Thr-592 phosphorylation in hemin-treated THP-1 cells. THP-1 cells were treated with 75 μM hemin for 24 hours. Cell lysates were analyzed and quantified as in panel A for 4 independent samples. (C) Restoration of hemin-mediated HIV-1 replication by knockdowns of SAMHD1. THP-1 cells were stably transduced with lentiviruses expressing SAMHD1-targeting shRNA and infected with HIV-1-LUC-G virus. Where indicated, the cells were treated with 75 μM hemin. Luciferase activity was measured 2 days PI. The mRNAs were measured by RT-PCR with 18S rRNA as an internal reference for ΔΔCt analysis. The means ± SD and P values calculated using Student t test are shown for 3 independent samples. (D) Hemin treatment increased SAMHD1 mRNA expression. THP-1 cells were treated with hemin for 24 hours. RNA was extracted, reverse transcribed, and analyzed by RT-PCR using primers for SAMHD1 and 18S rRNA as an internal reference for ΔΔCt analysis. The means ± SD and P values calculated using Student t test are shown for 3 independent samples. Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology

Protein network of iron-activated HIV-1 restriction factors in hemolytic anemia. Protein network of iron-activated HIV-1 restriction factors in hemolytic anemia. SCD condition or hemin treatment increases expression of HO-1 and FPN. FPN reduces intracellular iron levels stabilizing HIF-1α, and increasing IKBα and p21 production. It also decreases CDK2 expression and inhibits CDK2 activity. Decreased CDK2 activity reduces SAMHD1 phosphorylation and inhibits HIV-1 RT. Decreased CDK2 activity and increased IKBα expression can also inhibit HIV-1 transcription. Namita Kumari et al. Blood Adv 2016;1:170-183 ©2016 by American Society of Hematology