Heme Inhibits HIV-1 Through the Induction of Heme Oxygenase-1, Ferroportin, IKBα and p21
Namita Kumari
Center for Sickle Cell Disease, Howard University
OMICS Retroviruses and Novel Drugs, June 08-09, 2015
Two a helices each containing 233 residues with heme sandwiched in the center
Heme Oxygenase-1 (HO-1)
Heme Oxygenase-1 Pathway
Biliverdin BilirubinHeme
Fe2+
HO-1
+ 3 NADPH+ 3 H2O3 NADP+
NADPH NADP+
Fe2+
Heme Oxygenase-1 Inhibits HIV-1
Devadas and Dhawan, J. Immunol. 2006
Heme Processing by Tissue Macrophages
Beaumont C Haematologica 2010;95:1233-1236
©2010 by Ferrata Storti Foundation
Iron and HIV-1
• Increased iron stores correlated with faster HIV-1 progression (Gordeuk et al., 2001)
• Iron stimulates HIV-1 replication and DFO decreases it (Traore and Meyer, J. Clin Virol., Suppl. 2004)
• Iron chelators inhibit HIV-1 transcription (Debebe et al, Mol Pharmacol. 2011; Kumari et al., AAC 2014)
• Ferroportin inhibits and hepcidin induces HIV-1 transcription (Xu et al. Retrovirology 2010)
• Heme oxygenase-1 induction by hemin inhibits HIV-1 (Devadas and Dhawan, J. Immunol. 2006)
Iron HIF1α And p21
• Iron deprivation and oxygen deprivation (hypoxia) induce hypoxia-inducible factors, HIF1α (Chepleve et. al, 2011)
• Fe depletion induces universal cyclin- dependent kinase inhibitor, p21 (Richardson et. al, 2007)
Nekhai et al.
Regulation of HIV-1 Transcription by Iron and Hypoxia
Overexpression of p21 Downregulates NTP
Allouch et al., Proc. Natl Acad. Sci 2013
PPY (2-phenyl-1-pyridin-2yl-ethanone)-based Iron Chelators
Kumari et al., Antimicrob. Agents Chem. 2014
Bp4eT PPY PPYeT PPYaT
0 5 10 15 20 25 30 350
0.1
0.2
0.3
0.4
FeS+SIH
FeS
FeS+PPYeT
FeS+PPYaT
FeS+PPY
Time (min)
Flu
ores
cen
ce, (
F-F
o)/F
o (a
rbi-
trar
y u
nit
s)
DMSO PPY PPYaT PPYeT0
2
4
6
8
10
12
14
16
TF
R m
RN
A (f
old,
nor
mal
ized
to
18S
RN
A)
Cyclin A Cyclin E IkB0
2
4
6
8
10
12
DMSOPPYPPYaTPPYeT
Ge
ne
Exp
ress
ion
(F
old
, n
orm
aliz
ed
to b
-act
in)
PPY PPYaT pPYeT0
100
200
300
400
p2
1 E
xpre
ssio
n n
orm
aliz
ed
to
18
sr R
NA
pPY pPYaT pPYeT0
25
50
75
100
(32P
) P
hosp
hryl
atio
n (N
orm
aliz
ed t
o C
DK
2 E
xpre
ssio
n)
C
AB
histoneH1
PPY PPYaT PPYeT
CDK2
32PWB:
α –CDK2
Iron Chelators Inhibit CDK2 Activity and Induce Expression of p21 and IKBα
Kumari et al., Antimicrob. Agents Chem. 2014
PPY Iron Chelators Inhibit HIV-1 Transcription
mR
NA
(fo
ld,
no
rmal
ized
to
18S
RN
A)
0.0
0.5
1.0
1.5
2.0
2.5 UntreatedDMSOPPYPPYeTPPYaTAZT
mR
NA
(fo
ld,
no
rmal
ized
to
18S
RN
A)
Gag Env0.0
0.5
1.0
1.5
2.0 UntreatedDMSOPPYPPYeTPPYaT
**
*
Kumari et al., Antimicrob. Agents Chem. 2014
Heme Inhibits HIV-1 in THP-1 Cells and PBMC
Control Hm Hm+Hep Hep0
0.5
1
1.5
2
2.5
3 THP1 cells env gag
No
rma
lize
d E
xpre
ssio
n
Control Hm Hm+Hep Hep0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2 PBMCenv gag
No
rma
lize
d E
xpre
ssio
n
Control Hemin Hemin+Hepcidin Hepcidin0
500
1000
1500
2000
2500
Lu
cife
rase
Act
ivity
THP1-HIV-Luc
Control Hemin Hemin+Hepcidin Hepcidin0
50
100
150
200
250
300
350
400
450
500
Lu
cife
rase
Act
ivity
PBMC-HIV-Luc
Heme Induces HO-1, Ferroportin, p21 and IKBα
H0-1 Fpn1 p21 IKBα0
5
10
15
20
25
30 THP1 cells UT HEMIN
Nor
mal
ized
Exp
ress
ion
H0-1 Fpn1 p21 IKBα0
5
10
15
20
25PBMC UT HEMIN
Nor
mal
ized
Exp
ress
ion
Ferroportin Knockdown Alleviates HIV-1 inhibition by Hemin
THP1 Fpn1 sh RNA Control sh RNA0
20
40
60
80
100
120 UT HmL
uci
fera
se A
ctiv
ity (
% U
ntr
ea
ted
Co
ntr
ol)
THP1_G
ag
THP1FP
NKD_Gag
THP1sh
_Gag
THP1_E
ne
THP1FP
N1KD_E
ne
THP1sh
_Ene
0
0.4
0.8
1.2
1.6UT HM
No
rma
lize
d E
xpre
ssio
n
THP1 THP1Sh THP1 Hif1KD0
1000
2000
3000
4000
5000
6000
Luc
ifer
ase
acti
vity
HIF1α KD Induces One Round HIV-1 Infection
THP1 THP1Sh THP1-Hif1KD0
0.4
0.8
1.2
1.6
2Gag Env
Nor
mal
ised
Exp
ress
ion
Heme Treated THP1 cells In G1 Arrest
G0/G1 G2/M S0
10
20
30
40
50
60
70
80
90
UT Hemin
Cel
l Cyc
le(%
Tot
al)
UT Hemin0
1
2
3
4
5
6
7
IKB
α N
orm
aliz
ed
to
Tu
bu
lin
IKBα and NF-kB In Heme Treated Cells
Tubulin
IKBα
Nuclear Cytoplasmic
Control
Hemin
Control Hemin
Control-N
uclear
Control-C
ytoplas
mic
Hemin
-Nucle
ar
Hemin
-Cyto
plasm
ic0
0.4
0.8
1.2
NF
-kB
(p
65
)
Conclusions
• Hemin treatment induces ferroportin expression in THP-1 cells and PBMC
• Low intracellular iron mimics hypoxia as it upregulates and stabilizes HIF1α and p21
• Similar to hypoxia, iron depletion inhitis HIV-1
• Accumulation of inactive NF-kB in cytoplasm in hemin treated cells may inhibit HIV-1 transcription
Acknowledgement Center for Sickle Cell Disease Howard University Sergei Nekhai
Xiaomei NiuAndrey Ivanov
Viral Immunology Section, Food and Drug Administration Subhash Dhawan
NIH- NHLBI 1R01HL125005-01NIH -NHLBI 1P50HL118006-01NIH -NIMHD 5G12MD007597
Enamine, Ukraine: Dmytro Kovalskyy
George Mason University: Fatah Kashanchi Sergey Iordanskiy
Protein Network Activated in Macrophages by Hemolytic Anemia
HO-1Heme
Ferroportin
Fe2+Fe3+
Hepcidin
Fe2+
HO-1 FPN
Low Fe
HIF-1
Ischemia
Low O2
CDK9
S hemoglobin
p21, p27
HIV-1
Ceruloplasmin
CDK2
Hemolytic
anemia
PP1
Iron
chelators
inhibitors
Overexpression
Overexpression
inhibitors