BackgroundPhysiological regulation of cellular iron involves iron export by the membrane protein, ferroportin, the expression of which is induced by iron and negatively modulated by hepcidin. We previously showed that iron chelation is associated with decreased HIV-1 transcription. We hypothesized that increased iron export by ferroportin might be associated with decreased HIV-1 transcription, and degradation of ferroportin by hepcidin might in turn induce HIV-1 transcription and replication. Here, we analyzed the effect of ferroportin and hepcidin on HIV-1 transcription.ResultsExpression of ferroportin was associated with reduced HIV-1 transcription in 293T cells and addition of hepcidin to ferroportin-expressing cells counteracted this effect. Furthermore, exposure of promonocytic THP-1 cells to hepcidin was associated with decreased ferroportin expression, increased intracellular iron and induction of reporter luciferase gene expression. Finally, exposure of human primary macrophages and CD4+ T cells to hepcidin and iron was also associated with induction of viral production.ConclusionOur results suggest that the interplay between ferroportin-mediated iron export and hepcidin-mediated degradation of ferroportin might play a role in the regulation of HIV-1 transcription and may be important for understanding of HIV-1 pathogenesis.

HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-κB, and others. The cells of most of the body’s organs are exposed to ~3–6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O2, and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O2 compared to 21% O2. HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O2 compared to 21% O2. This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O2, the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-κB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O2 and also expression of CDK9/cyclin T1-dependent IκB inhibitor α was repressed. Our results suggest that lower HIV-1 transcription at 3% O2 compared to 21% O2 may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O2 and that additional host cell factors such as CDK2 and NF-κB might be major regulators of HIV-1 transcription at low O2 concentrations.

ciency or ferroportin Q248H had lower circulating tumor necrosis factor-α concentrations than iron-replete children with WT ferroportin, 25 led us to hypothesize that ferroportin Q248H has reduced sensitivity to hepcidin but that this property would be observed at lower concentrations of hepcidin than those used in previous studies. 23 Here, we analyzed the sensitivity of ferroportin Q248H to hepcidin at various concentrations by determining the levels of ferroportin transiently expressed in cultured cells and in human primary monocytes derived from humans with different ferroportin genotypes. We also measured ferritin concentrations in these cells, levels that are indicative of cellular iron status. Finally, we examined the effect of Q248H on serum iron measures in patients with sickle cell anemia who have markedly increased macrophage iron export because of chronic hemolysis. Design and Methods Predictive analysis and worldwide allele frequenciesMinor allele frequencies for missense mutations in the single nucleotide polymorphism database (dbSNP) were determined using Kaviar and sequence data (http://db.systemsbiology.net/kaviar). 26Mutations were analyzed for effects on protein function using a 571-residue ferroportin protein (UniProtKB/Swiss-Prot Q9NP59) and the predictive analysis tools: SIFT (Sorting Intolerant From Tolerant; http://blocks.fhcrc.org/sift/SIFT.html) 27 and PolyPhen2 (Polymorphism Phenotyping version 2; using HumDiv model; http://genetics.bwh.harvard.edu/pph2/index.shtml). 28 Cells and media293T cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies) and 1% glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C in the presence of 5% CO 2 . Ferroportin expression constructsHuman WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants cloned with c-Myc and histidine tags in a pcDNA3.1 expression vector were kindly provided by Dr. Hal Drakesmith. 23 To generate enhanced green fluorescent protein (EGFP)-tagged human ferroportin, the ferroportin coding sequences from WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants were amplified by polymerase chain reaction (PCR) with the following primers that included XhoI and Kpn1 restriction sites (shown in italics): forward primer, GCCTC-GAGATGACCAGGGCGGGAGATCAC and reverse primer, GCGGTACCGTAACAACAGATGTATTTGCTTGATTTTC. The PCR products were purified on agarose gel, digested with XhoI and Kpn1 (BioLabs, Ipswich, MA) and ligated into the pEGFP-N1 vector (Clontech, Mountain View, CA, USA) which was also digested with XhoI and Kpn1 and ligated. The ligation products were transformed into E. coli DH5α cells (Invitrogen) and kanamycin-resistant colonies were selected. WT ferroportin-EGFP, C326Y and Q248H ferroportin -GFP-expressing plasmids were purified using a Qiagen (Valencia, CA, USA) purification kit and sequenced using the Macrogen service (Rockville, MD, USA). Transfection and treatment293T cells were seeded in 6-well ...

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) pose major public health concerns worldwide. HCV is clearly associated with the occurrence of hepatocellular carcinoma, and recently HIV infection has also been linked to the development of a multitude of cancers. Previously, we identified a novel nucleoside analog transcriptional inhibitor ARC (4-amino-6-hydrazino-7-b-mide) that exhibited proapoptotic and antiangiogenic properties in vitro. Here, we evaluated the effect of ARC on HIV-1 transcription and HCV replication. Using reporter assays, we found that ARC inhibited HIV-1 Tatbased transactivation in different cell systems. Also, using hepatoma cells that harbor subgenomic and full-length replicons of HCV, we found that ARC inhibited HCV replication. Together, our data indicate that ARC could be a promising candidate for the development of antiviral therapeutics against HIV and HCV.

HIV‐1 transcription is induced by viral transcriptional activator, Tat protein that recruits transcriptional co‐activators, including CDK9/cyclin T1 to the HIV‐1 promoter. We recently showed that Tat is a substrate for CDK2 and that mutations in the Ser16 and Ser 46 residues of Tat that were phosphorylated by CDK2, prevented HIV‐1 transcription and viral replication. We also recently showed that iron chelators ICL670 and 311 inhibited on Tat‐induced HIV‐1 transcription by inhibiting cellular activities of CDK2 and CDK9. Thus our previous studies suggest that a decrease in cellular iron might have a protective effect against HIV‐1. Here, we analyzed the effect of iron exporter, ferroportin on HIV‐1 transcription and viral replication. Expression of ferroportin in 293T cells dramatically inhibited HIV‐1 transcription. Induction of ferroportin expression in THP‐1 cells inhibited one round of replication of pseudotyped HIV‐1. Previously, ferroportin Q248H mutation was shown to have a potential protective effect against iron deficiency in children exposed to repeated inflammatory conditions. We analyzed the effect of hepcidin on the expression of ferroportin and found that the Q248H mutant was less sensitive to lower concentrations of hepcidin. This finding suggested that Q248H mutation might be protective against HIV‐1 infection under conditions of mild inflammation. We are currently investigating the direct effect of hepcidin and ferroportin Q248H mutation on HIV‐1 transcription.