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BioCIHP Meeting, Cochin Institute 23rd, April 2012. Nicolas Roy 3eme année de Doctorat. BST2/Tetherin: a restriction factor of HIV-1. Implication of the ubiquitination pathway in its downregulation by the viral protein Vpu. Laboratory director : Dr. Clarisse Berlioz-Torrent - PowerPoint PPT Presentation
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BST2/Tetherin: a restriction factor of HIV-1. Implication of the ubiquitination pathway in its downregulation by the
viral protein Vpu.
Nicolas Roy3eme année de Doctorat
BioCIHP Meeting, Cochin Institute
23rd, April 2012
Laboratory director : Dr. Clarisse Berlioz-TorrentThesis supervisor : Dr. Katy Janvier
Laboratoire « Interactions Hôte-Virus »Institut Cochin
INSERM U1016 – CNRS UMR 8104 Département «3I»
• The Human Immunodeficiency virus (HIV) is the causal agent responsible for the Acquired Immuno-deficiency syndrome or AIDS
• Globally, 34.0 million people are living with HIV.
• 1.7 million people worldwide died from AIDS‑related causes, down 24% from the peak in 2005
Public Health and HIV
(Report on the world AIDS epidemic agency, ONUSIDA, 2012)
HIV-1 replication cycle
HIV-1
CD4
Co-receptor
EntryReverse-transcription
Integration
Transcription
ARN
Translation
HIV-1
Assembly
Gag
Gag-Pol
Enveloppe glycoprotein
Release
Early steps Late steps
Molecular and cellular mechanisms involved in the release of HIV-1 particles by Vpu
Vpu
R U5 GAG
POL
VifVpr Vpu ENV Nef
R U3Tat
RevGenome of HIV-1
Vpu
Vpu is an accessory protein
Vpu is a small transmembrane protein (16kDa)
Vpu is encoded by HIV-1 and some strains of SIV
Vpu: an accessory protein of HIV-1
Vpu: an accessory protein of HIV-1
From Perez-Caballero et coll, Cell, 2009
BST2
From Bienasz et coll, Cell Host and Microbe, 2009
Vpu is involved in CD4 degradation and in the release of HIV-1 particles.
Vandamme et coll, Cell Host and Microbe, 2008
Miyagi et coll, PNAS, 2009
Decrease of BST2 cell surface expression Targeting of BST2 for degradation
The protein BST2 tethers mature viral particles at the plasma membrane (Perrez-Caballero et coll, Cell, 2009)
Vpu promotes an efficient release of viral particles by counteracting the
cellular protein BST2, recently described as a restriction factor of HIV release (Vandamme et coll, 2008; Neil et coll, 2008)
Vpu restores HIV release through down-regulation of BST2 expression
Infected cells (Vpu-) Infected cells(Vpu+)Release -/+ Release +++
The modulation of BST2 expression induced by Vpu restores an efficient release of viral particles
The molecular mechanisms involved in this process remain to be deciphered.
HRS (ESCRT-0) is required for an efficient release of HIV-1 particles
Implication of the ubiquitination pathway in BST2 downregulation by Vpu
Recycling decreasedEndocytosis through AP-2
Recruitment by the ESCRT machinery
Lysosome
Late endosomesMVB
Decreasing of BST2 level on the cell
Infected cells (Vpu+)
+ Vpu
Endosome
ESCRT
HRSUb
Increased rate of BST2 degradationFrom Stenmark et coll, Nature, 2009
Janvier et coll., PloS Pathogen, 2011
BST2 is ubiquitinated and Vpu increases its ubiquitination
HA-Ub
Flag-BST2
Flag-BST2
HA-Ub + + + +- - - -
- - + + - - + +
Lysates Flag-IP
HA-Ub
Flag-BST2
Flag-BST2HA-Ub
+ + + + + + + +- -+ + - -+ +
NL4.3 W
TNL4.3
Udel
NL4.3 W
TNL4.3
Udel
Lysates Flag-IP
E3 ligases : siRNA screening
E1 ATPUb E2Ub+ E1
E2
Ub
Substrat
Ub
E3
E3
Ub K
UbUb
K
KKUb
siRNA Screening
HECT E3-ligases:
Nedd4
Nedd4-L
WWP1
ITCH
RING E3-ligases:
RNF189
RNF138
CBL
β-TrCp ½
UBAP1
Role of the E3 ligase on BST2-regulation in a non-infectious context
Role of the E3 ligase on BST2-regulation in an infectious context
Effect of E3-ligases depletion on BST2 expression level
HeLa
siRNA E3 ligase
J1
Western blot analysis
Cell lysis
J5
- siC
T- s
iNed
d4- s
iNed
d4-L
- siR
NF18
9- s
iCBL
- siIT
CH
- siW
wp1
- siβ
-TrC
p
- siR
NF13
8- s
iUBA
P1
BST2
Tubuline
siCD
-siN
edd4
-siN
edd4
-L -
siRN
F189
-siC
BL -siI
TCH
-siW
wp1
-siR
NF1
38 -
siβ-T
rCp
-siU
BAP1
-
BS
T2
exp
ress
ion
leve
l(%
of
siC
D)
Western-blot analyses of E3 depletions
RT-PCR analyses of depletions
Western-blot analyses of BST2 expression
Odyssee quantification
Nedd4 -
Nedd4-L -
CBL -
ITCH -
Wwp1 -
UBAP1 -
- siC
T- s
iNed
d4- s
iNed
d4-L
- siR
NF18
9- s
iCBL
- siIT
CH
- siW
wp1
- siβ
-TrC
p
- siR
NF13
8- s
iUBA
P1
β-TrCp2 -
- si β
-TrC
p2
- siC
T
β-TrCp1 -
- siC
T
RNF189 -
- si R
NF189
- siC
T
- si β
-TrC
p1
Effect of E3-ligases depletion on BST2 cellular localization
BST2 BST2 BST2 BST2
HRS HRS HRS HRS
BST2 HRS BST2 HRS BST2 HRS BST2 HRS
si CT si Nedd4 si RNF189 si β-TrCp
siRNA E3 ligase
J1
J5
Immunofluorescence microscopy
HeLa
RNF189 depletion leads to the accumulation of BST2 in HRS-positive compartments
Interaction of BST2 with E3 ligases
HeLa Flag-BST2
BST2 interacts with Nedd4 and RNF189
HA-Nedd4 -
Flag-BST2 {
Flag-BST2: +- +-
Lysates IP
HA-RNF189 -
Flag-BST2 {
Flag-BST2: +- +-
Lysates IP
HA-WWP1 -
Flag-BST2 {
Flag-BST2: +- +-
Lysates IP
HA-HRS -
Flag-BST2 {
Flag-BST2: +- +-
Lysates IP
pHA-E3 ligase
24h
Cell lysis
Flag pull-down
Western blot analysis
J1
J2
HA-βTrCp -
Flag-BST2 {
Flag-BST2: +- +-
Lysates IP
Co-immunoprecipitation analyses
Nedd4 and RNF189 are involved in BST2 ubiquitination
HeLa Flag-BST2
Cell lysis
Flag pull-down
Western blot analysis
J1
J4
siRNA E3 ligase
HA-Ub
J5
- siC
T- s
iNed
d4- s
iNed
d4-L
- siR
NF18
9
- siC
BL- s
iITC
H- s
iWw
p1- s
iβ-T
rCp
- siC
T
Nedd4 -
Nedd4-L -
CBL -
ITCH -
Wwp1 -
Effect of E3-ligases depletion on BST2 ubiquitination
Ubiquitination assay Western-blot analyses of E3 depletions
- siC
T- s
iNed
d4- s
iNed
d4-L
- siR
NF18
9
- siC
BL- s
iITC
H- s
iWw
p1- s
iβ-T
rCp
- siC
T
- siC
T
- siN
edd4
- siN
edd4
-L- s
iRN
F189
- siC
BL- s
iITC
H- s
iWw
p1- s
iβ-T
rCp
- siC
T
HA-Ub
Flag-BST2
HA-Ub - + + + + + + + + - + + + + + + + +
Lysates Flag-IP
Effects of E3-ligases depletion on BST2 turnover
Cycloheximide chase: Western-blot analysessiRNA E3 ligase
HeLaJ1
Western blot analysis
Cell lysis
CHX Chase
J5
Nedd4 & RNF189 regulate BST2 turn-over
CHX (h) 0 ¼ ½ 1 2 4 0 ¼ ½ 1 2 4 0 ¼ ½ 1 2 4 0 ¼ ½ 1 2 4
siCD siNedd4 siRNF189 siβTrCp
BST2
Tubuline
Conclusions (1)
- In a non-infectious context
Nedd4 & RNF189 are involved in BST2 turnover
Nedd4 & RNF189 are involved in BST2 ubiquitination
E3 ligases : siRNA screening
E1 ATPUb E2Ub+ E1
E2
Ub
Substrat
Ub
E3
E3
Ub K
UbUb
K
KKUb
siRNA Screening
HECT E3-ligases:
Nedd4
Nedd4-L
WWP1
ITCH
RING E3-ligases:
RNF189
RNF138
CBL
β-TrCp
UBAP1
Role of the E3 ligase on BST2-regulation in a non-infectious context
Role of the E3 ligase on BST2-regulation in an infectious context
Effects of E3-ligases depletion on Vpu-induced BST2 down-regulation
BST2 BST2 BST2 BST2
Env
BST2 BST2 BST2 BST2
Env Env Env
Env Env Env Env
si CT si Nedd4 si RNF189 si β-TrCp
siRNA E3-ligase
HeLaJ1
Immunofluorescence microscopy
HIV (NL4.3 WT)
J3
J5
Nedd4 &RNF189 are not involved in Vpu-induced targeting of BST2 for degradation
But, β-TrCp is required for this activity
Effects of E3-ligases depletion on viral production
siRNA E3-ligases Viral production
HIV-1 (NL4.3 VSV-G)
24h48h
HeLa CAp24 quantification
WB
Infectivity
Neither RNF189 nor β-TrCp are required for HIV-1 release
Conclusions (2)
Nedd4 & RNF189 do not seem to be required in Vpu-induced downregulation of BST2
Interestingly the E3 ligase β-TrCp is necessary for BST2 down-regulation by the viral protein Vpu
In a non-infectious context:
In an infectious context:
Neither for HIV-1 release
Nedd4 & RNF189 are involved in the regulation of BST2 ubiquitination and turnover
HOWEVER,
Vpu does not hijack cellular protein naturally involved in BST2 regulation but rather use another protein with a similar function
Katy Janvier Clarisse Berlioz-Torrent
Sophie LambertStéphane Frémont
Grégory PaciniUrsula Madjo
Sophie AbélanetVanessa Gourhand
Clarisse Berlioz-Torrent’s group
Platforms
Microscopy Sequencing
Flow Cytometry
Stephane Emiliani’s group
Supplementary data
HRS and E3-ligases localisation
RNF189RNF189 RNF189
Effect of E3-ligases overexpression on BST2 expression
Nedd4 and RNF189 overexpression lead to a decrease of expression of BST2
GFP-E3 Ligases
J1
Immunofluorescence microscopy
J3
J2
HeLa
BST2 BST2 BST2 BST2 BST2
Vpu Nedd4 RNF189 βTrCp
BST2 BST2 BST2 BST2 BST2Vpu Nedd4 RNF189 βTrCp
Effect of E3-ligases overexpression on cell-surface BST2 expression
Nedd4 and RNF189 overexpression lead to a decrease of cell-surface expression of BST2
pHA-E3 Ligases
J1
Immunofluorescence microscopy
J3
J2
HeLa
Effect of E3-ligases overexpression on cell-surface BST2 expression
pHA-E3 Ligases
J1
Immunofluorescence microscopy
J3
J2
HeLa
RNF189 RNF189 CS βTrCp βTrCp ΔF
RNF189 RNF189 CS βTrCp βTrCp ΔF
BST2 BST2 BST2 BST2
BST2 BST2 BST2 BST2
BST2 BST2 BST2 BST2
BST2 BST2 BST2 BST2
Vpu Vpu 2.6 Nedd4 Nedd4 CS
Vpu Vpu 2.6 Nedd4 Nedd4 CS
siRNA E3-ligase
HeLaJ1
Western blotCAp24 ELISA quantification
Infectivity
Lyse
NL4.3 WTorNL4.3 Udel
J3
J5
* *
* *
BST2 {
GP160 -GP120 -
GP41 -
Pr55GAG -
CAp24 -
Tubuline -
- NI
- si C
T- s
i Ned
d4- s
i Ned
d4-L
- si R
NF1
89- s
i CB
L- s
i CT
- si N
edd4
- si R
NF1
89- s
i CB
L
- si N
edd4
-L
- NI
- si C
T- s
i IT
CH
- si β
-TrC
p- s
i WW
P1- s
i CT
- si I
TC
H- s
i β-T
rCp
- si W
WP1
NL4.3 UdelNL4.3 WT NL4.3 UdelNL4.3 WT
RNF189 is not involved in Vpu-induced targeting of BST2 for degradation
Effects of E3-ligases depletion on viral production
Effects of E3-ligase depletion on Vpu-induced cell surface BST2 down-regulation
HeLa
RNF189 is not involved in Vpu-induced BST2 cell-surface downregulation
But, β-TrCp is required for this downregulation
siRNA E3 ligase
J1
Flow cytometry
HIV (NL4.3 WT)
J3
J5
si CT si Nedd4 si RNF189
si β-TrCp si WWP1
Env + Env + Env +
Env + Env +
BS
T2
Env
uninfected
infected
uninfected
infected
uninfected
infected
uninfected
infected
uninfected
infected
Effects of E3-ligases depletion on BST2 turnover
siRNA E3 ligase
HeLaJ1
Western blot analysis
Cell lysis
CHX Chase
Cycloheximide chase: Western-blot analysis
J5
Nedd4 regulates BST2 turn-over
0 0,5 1 2 4CHX (h) 0 0,5 1 2 4 0 0,5 1 2 4 0 0,5 1 2 4
si CD si Nedd4 si RNF189 siβ-TrCp
BST2
Tubuline -
0
20
40
60
80
100
120
0 50 100 150 200 250 300
Time (min)
BS
T2
exp
ress
ion
leve
l (
% o
f T
=0) siCD
siNedd4
siRNF189
siβTrCp
