HIV-1 VPR causes G2 arrest and apoptosis via DNA replication stress

Erik Zimmerman

Vicente Planelles Lab

Vpr functions

PIC import

G2 arrest

G2 arrest enhances proviral transcriptionand translation

Abbas, Cell. and Mol. Immunology, 2003

Vpr

ATR Rad17 Rad9-Hus1-Rad1

?

Apoptosis

BRCA1

P

GADD45Cdc25C

Checkpoint activation

Chk1

G2 M

Cdk1/cyclin B

P

P

P

P

Active ATR complexP

1. Is ATR activated in the context of HIV-1 infection?

2. What is the nature of the ATR-activating stimulus?

3. Is transition to G2 requisite for apoptosis?

ATR mediates G2 arrest and apoptosis

ATR is required for HIVNL4-3 induced G2 arrest in primary CD4+ lymphocytes

HIV-infected CD4+ T cells are G2 arrested in vivo

• PBMC isolated from recent seroconverters prior to initiation of HAART

• Stained for CD4, p24 Gag, and DNA content

Conclusions #1

• ATR is essential for HIV-1 induced G2 arrest

• HIV-1 infected cells are arrested in G2 in vivo

What is the ATR-activating stimulus?

Adapted from Zou and Elledge. Science, 2003

RPA (ssDBP)

ATR + ATRIP

Rad17 complex + 9-1-1

DNA polymerase complex

RPA 17RPA 32-PRPA 70

A model for ATR Activation

Can we detect RPA accumulation in HIV-infected cells?

HIV-1 VPR induces RPA-rich nuclear foci in primary CD4+ cells

P24 Gag

RPA32

DIC

Mock HU NL4-3 NL4-3 VprX

HIV-1 VPR induces RPA-rich nuclear foci

0

10

20

30

40

50

60

70

80

90

100

Uninfected

HU AphNL4-3+

NL4-3 VprX+

AD8+

AD8 VprX+

% of cells with RPA-rich foci

Conclusions #2

• VPR causes DNA replication stress, accumulation of RPA-rich foci in primary CD4+ lymphocytes

• Activation of ATR by VPR may require DNA replication

Can we prevent the cytopathic consequences of VPR by prohibiting DNA replication/entry into G2?

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200

Thym

Channels (FL2-A-DNA (FL2-Area))0 40 80 120 160 200

Vpr + Thym

48 hrs

Channels0 30 60 90 120 150

2mM Thym

6 hr

12hr

18 hr

24 hr

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

Tim

e p

ost

-th

ymid

ine

rel

eas

e

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

Channels (FL2-A-DNA (FL2-Area))0 50 100 150 200 250

What about post-mitotic HIV-1 target cells?

Cycling into G2 is required for VPR-induced apoptosis

Two different HIV target cells:

• Activated CD4+ T cells– Dividing– HIV-infection is highly

cytopathic– ~1.6 day half-life– VPR causes G2 arrest,

apoptosis

• Macrophages– Nondividing– Highly resistant to HIV-

induced apoptosis– half-life of several days– VPR enhances PIC

import

Is the ATR pathway present in nondividing cells?

ATR is expressed only in dividing cells

Conclusions• VPR induces an ATR-dependent G2 arrest in primary

CD4+ T cells

• p24+ CD4+ lymphocytes from recent seroconverters are arrested in G2 in vivo

• VPR causes accumulation of RPA-rich nuclear foci, indicative of DNA replication fork stalling

• VPR cannot activate ATR or induce apoptosis in non-cycling cells (G1 blocked Hela or macrophages)– Could this help explain the resistance of macrophages to HIV-1

induced apoptosis?– Can we sensitize quiescent, nondividing reservoirs to the pro-

apoptotic effects of VPR?

Acknowledgements

VPR expression causes accumulation of RPA-rich nuclear foci in transduced Hela cells

Do these observations apply in vivo?-primary CD4+ lymphocytes-physiological levels of VPR-presence of other viral gene products

Mock HU pHR-GFP pHR-VPR

RPA-32

GFP

ATR is required for VPR-induced G2 arrest

Mock pHR-GFP pHR-VPR

G2=47%

G2=88%

G2=90%

Vpr fails to activate ATR in primary macrophages

Mock IR pHR-GFP pHR-VPR

DIC

-H2AX

GFP