HIV-1 VPR causes G2 arrest and apoptosis via DNA replication stress
-
Upload
aphrodite-harrington -
Category
Documents
-
view
41 -
download
0
description
Transcript of HIV-1 VPR causes G2 arrest and apoptosis via DNA replication stress
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