Éric a. cohen laboratory of human rétrovirology ircm
DESCRIPTION
HIV-1 Vpr-mediated ATR activation and G2 cell-cycle arrest induce up-regulation of NKG2D ligands and activate NK cell-mediated killing. Éric A. Cohen Laboratory of Human Rétrovirology IRCM 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Cape Town July, 20, 2009. - PowerPoint PPT PresentationTRANSCRIPT
HIV-1 Vpr-mediated ATR activation and G2 cell-cycle arrest induce up-regulation of NKG2D ligands and
activate NK cell-mediated killing
Éric A. Cohen Laboratory of Human
RétrovirologyIRCM
5th IAS Conference on HIV Pathogenesis, Treatment
and Prevention,Cape Town
July, 20, 2009
Viral Protein R (Vpr)
• HIV accessory protein of 96 aa (14 kDa)
• Exists in 3 different forms- Virion-associated (~275
molecules/virion)
- Intracellular forms- Extracellular forms:
Plasma and CSF
• Displays cell transduction properties
Vpr Biological Activities
• Facilitates infection of nondividing cells such as terminally differentiated macrophages
• Causes cell-cycle arrest at the G2 phase in CD4+ T cells via activation of the ATR-mediated DNA damage/stress signaling pathway – Conserved among HIV and SIV Vpr
– Abnormal accumulation of infected cells in G2 in HIV- infected subjects
How Does Vpr Induce a G2 Arrest?
ATR
Chk1
Cdc2
Cyclin B
Cdc2
Cyclin B
?
ATM
DNA stress/damage checkpoint G2 arrest
Belzile et al., PLoS Pathogens, 2007
Functional Relevance of Vpr-induced G2 Arrest
• Vpr-induced G2 arrest has two downstream effects:– Enhances proviral transcription.– Promotes apoptosis of infected cells– Other Effects?
• Activation of the DNA damage/stress checkpoint pathway initiated by ATR or ATM kinase upregulates ligands of the activating NKG2D receptor and enhances susceptibility of target cells to NK cell killing (Gasser et al., 2005)
ATRNKG2D
Control of NK Cell Effector Function
• under control of a sophisticated and tightly regulated network of activating and inhibitory receptors
• NKG2D is a central activating receptor that modulates NK cell function – MHC class 1-related chains A/B
(MIC A/B)– UL16-binding protein (ULBP-1,
-2, -3, -4)
• NKG2D is expressed not only on NK cells, but also on T cells, CD8+T- cells, and a subset of CD4+ T-cells
• Expression of NKG2D ligands is largely confined to virus-infected, tumor and stressed cells.
RATIONALE
HIV-1 infection of primary CD4+ T-lymphocytes increases cell-surface expression of specific NKG2D ligands both in vitro (Cerboni et al. , 2007;
Ward et al. 2007) and in vivo (Fogli et al., 2008)
GOAL
To investigate whether expression of Vpr could regulate cell-surface expression of NKG2D
ligands and modulate NK cell cytotoxic activity
HIV-1 upregulates cell-surface expression of ULBP-2 in primary CD4+ T-cells in a
Vpr-dependent manner
PBMCs of healthy
subjects
Infection MOI= 0.5(GFP-marked R5 virus)
WT/ΔVpr-
Cell-surface staining anti-NKG2D ligands
on GFP+ cells
4-5 daysPrimary CD4+ T-lymphocytesactivated with
PHA/IL2
No upregulation of MICA and MICB cell-surface expression
N=5
ULBP-1 ULBP-2 ULBP-3
Rel
ativ
e ce
ll nu
mbe
r
Mock: 2.83
HIVΔVpr: 3.23
HIV WT: 2.96
Mock: 23.91
HIVΔVpr: 23.84
HIV WT: 40.65
Mock: 7.27
HIVΔVpr: 7.89
HIV WT: 7.38
Vpr enhances susceptibility of R5 HIV-1-infected-CD4+ T-lymphocytes to NK cell-mediated killing
Infected cells (GFP+) were exposed to autologous non-activated primary NK cells in a 4h-51Cr release assay
Vpr promotes NK cell-mediated killing at least in part via NKG2D
N=3
Donor 1 Donor 2
Expression of Vpr alone is sufficient to upregulate ULBP2 cell-surface expression
N=2
ULBP2
WPI-Vpr WT 126.9
WPI 45.4
Cel
l rel
ativ
e n
um
ber
Lentiviral vectors expressing GFP
WPI
WPI-Vpr WT
Primary CD4+ T-lymphocytes
Vpr, in the absence of any other HIV proteins, upregulates NKG2D ligands non-selectively
suggest that perhaps other viral protein(s) may limit cell-surface expression of some ligands
Lentiviral vector-transduced (GFP+) CEM.NKR T cells
ULBP-1 ULBP-2 ULBP-3 MICA MICB
Rel
ativ
e ce
ll n
um
ber
WPI-Vpr WT 58.2WPI
25.9
WPI-Vpr WT 97.8WPI
54.5
WPI-Vpr WT 28.6WPI 9.0
WPI-Vpr WT 315.8
WPI 82.8
WPI-Vpr WT 311.6
WPI174.9
APC 41.6
Mock29.4
APC 95.1
Mock52.1
APC 20.3
Mock8.7
APC 427.1Mock
91.7
APC 354.9Mock125.2
Upregulation of NKG2D ligands is dependent on Vpr-mediated G2-checkpoint arrest
R80A
Q65R
CEM.NKR T cells
WPI : 118.7WPI-Vpr R80A : 126.2WPI-Vpr Q65R : 200.1WPI-Vpr WT : 358.1
ULBP-2
Rel
ativ
e ce
ll n
um
ber
A
B
Vpr-mediated upregulation of NKG2D ligands is inhibited by Caffeine
CEM.NKR T cells ULBP-2
Rel
ativ
e ce
ll n
um
ber
WPI-Vpr WT 31.7
WPI 21.1
WPI-Vpr WT 102.5
WPI 35.1
APC 48.4
Mock24.8
APC 327.4
Mock 45.0
+ CaffeineNot treated
A B
Upregulation of NKG2D ligands by Vpr triggers NK cell cytotoxic responses
Transduced cells populations were exposed to non-activated primary NK cells in a 4h-51Cr release assay
CEM.NKR T cells N=2
BA
Delivery of virion-associated Vpr via defective HIV-1 particles upregulates ULBP-2 in non-infected target cells
N=3
Indinavir-treated HIVΔVpr HIVΔVprΔRTΔIN
Donor 1 Donor 2
Indinavir-treated HIVΔVpr
A B C
D
+ Vpr WT46.0
+ Vpr Q65R14.8
+ Vpr WT35.5
+ Vpr Q65R17.8
Conclusions• HIV upregulates selectively cell-surface expression of ULBP-2 in
primary CD4+ T-cells in a Vpr-dependent manner
• Vpr alone is sufficient to upregulate expression of NKG2D ligands, including ULBP-1, -2, -3, MICA and MICB, suggesting that HIV replication and expression of other virus proteins are not required for this activity of Vpr
• The absence of selective NKG2D ligand upregulation when Vpr is expressed alone suggests that another viral factor limits cell-surface expression of some ligands during HIV-1 infection
• Vpr upregulates expression of NKG2D ligands by a process that relies on the recruitment of the DDB1-CUL4A (DCAF1) E3 ligase and on activation of the ATR/ATM-mediated DNA damage/stress checkpoint pathway
Vpr is a key viral determinant responsible for induction of NKG2D ligands during HIV-1 infection
Conclusions
• Vpr expressed alone or during HIV-1 infection promotes NKG2D-dependent NK cell cytotoxic responses via its ability to upregulate NKG2D ligands
• Delivery of virion-associated Vpr via HIV-1 defective particles induces analogous effects in non-infected target cells, suggesting that Vpr may act similarly beyond infected cells
Overall, this study suggests an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced
CD4+ T lymphocyte depletion but may also play a part in HIV-1-induced NK cell dysfunction through sustained
NKG2D receptor activation
Acknowledgements
Dr. D. Trono (EPFL, Lausanne) Dr. H. Göttlinger (U. of Mass.) Dr. Y. Ishizaka (Tokyo)
ReagentsLaboratory of Human Retrovirology
RESEARCH TEAM IN HIV PATHOGENESIS (HPAT)
JP BelzileJ. Richard
S. Sindhu
Flow Cytometry
• Eric Massicote (IRCM)• Martine Dupuis (IRCM)
IRCM Clinic
• Dr P. Larochelle• Mrs M. Gauthier
All the volunteers
Expression of Vpr WT and Vpr mutants (R80A and Q65R) in CEM.NKR T cells
WPI : 37.9WPI-Vpr R80A : 113.0WPI-Vpr Q65R : 145.5WPI-Vpr WT : 111.4
Rel
ativ
e ce
ll n
um
ber
Vpr
ULBP-2
Rel
ati
ve
cel
l n
um
ber
GFP + GFP -
WPI-Vpr WT 49.5WPI28.1
WPI-Vpr WT 80.6WPI31.2