Heme Oxygenase-1, Ferroportin and SAMHD1 Induction By Heme Inhibits HIV-1

Background

Heme oxygenase-1 (HO-1) induction by hemin inhibits HIV-1 infection in cultured macrophages and T-cells and also in HIV-1 infected humanized mice via a protein kinase C-dependent pathway (reviewed in [1]). LPS-treated human macrophages express HO-1 which may protect them against HIV-1 infection through the production of MIP1α, MIP1β and LD78β chemokines that decrease CCR5 expression. Iron depletion by iron chelators or through the expression of ferroportin, an iron export protein, inhibited HIV-1 [2, 3]. 

Objective

We analyzed the effect of heme on HIV-1 infection in macrophages and the contribution of heme  and iron regulatory proteins including HO-1, ferroportin and hepcidin and cell cycle regulatory cell cycle dependent kinase 2 (CDK2) and p21, which are deregulated by iron depletion. We also analyzed SAM domain and HD domain-containing protein 1 (SAMHD1) as it was shown to be regulated by CDK2 and p21.

Methods

One round HIV-1 infection was analyzed in THP-1 cells infected with VSVg-pseudotyped HIV-1 expressing luciferase. Gene expression and protein expression analysis was carried out using RNA and protein isolated from hemin treated and untreated cells using RT PCR western blot respectively. Specific gene knock-downs were generated in THP-1 cells using lentivirus expressing gene-targeted shRNAs.

Results

In heme-treated THP-1 cells, mRNA expression of ferroportin, p21, SAMHD1, hypoxia-induced factor (HIF)-1α, and IKBα and NF-κB inhibitor was increased and CDK2 expression decreased. Stable ferroportin knock-down prevented HIV-1 inhibition in THP-1 cells. Treatment with hepcidin also restored HIV-1 inhibition in hemin treated THP-1 cells. Ferritin levels increased in Hemin treated THP1 cells and decreased when cells were treated with tin protoporphyrin (SnPP) IX inaddtion to hemin, suggesting that HO-1 induction leads to intracellular iron accumulation. In addition, hemin treatment reduced the expression of p65 subunit of NF-kB and induced expression of NF-κB inhibitor, IKBα. We also observed increased expression of p21 and decreased expression of   CDK2 in the cells treated with hemin and accumulation of the cells in the G1 phase of the cell cycle. Both CDK2 and p21 control phosphorylation of SAMHD1 which in turn controls the dNTP pool and prevents HIV-1 reverse transcription. We observed reduced expression of CDK2 and increased expression of SAMHD1 and p21 in hemin treated cells, which may also contribute to the inhibition of HIV-1. Stable HIF-1a knockdown in THP-1 cells increased HIV-1 replication indicating that HIF-1a might also restrict HIV-1 replication.

Conclusion

Our study shows that induction of HIF-1a and iron export and utilization proteins protects hemin-treated THP-1 cells from HIV-1 infection.  Block of HIV-1 inhibition by heme in THP-1 cells with stable ferroportin knock-down and restoration of  HIV-1 replication with hepcidin in heme-treated THP-1 cells suggest that ferroportin plays a key role in the HIV-1 inhibition. Additional molecular mechanisms of heme-mediated HIV-1 inhibition might also include NF-kB inhibition by IKBα,  reduction of CDK2 expression and induction of SAMHD1 and p21. Thus this complex deregulation of iron metabolism leads to the inhibition of HIV-1 transcription.

Acknowledgments

This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, U19AI109664 and 5G12MD007597) and District of Columbia Developmental Center for AIDS Research grant (1P30AI117970). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References

1.   Nekhai S, Kumari N, Dhawan S: Role of cellular iron and oxygen in the regulation of HIV-1 infection. Future Virol 2013, 8(3):301-311.

2.   Kumari N, Iordanskiy S, Kovalskyy D, Breuer D, Niu X, Lin X, Xu M, Gavrilenko K, Kashanchi F, Dhawan S et al: Phenyl-1-Pyridin-2yl-ethanone-based iron chelators increase IkappaB-alpha expression, modulate CDK2 and CDK9 activities, and inhibit HIV-1 transcription. Antimicrob Agents Chemother 2014, 58(11):6558-6571.

3.   Xu M, Kashanchi F, Foster A, Rotimi J, Turner W, Gordeuk VR, Nekhai S: Hepcidin induces HIV-1 transcription inhibited by ferroportin. Retrovirology 2010, 7:104.