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