BST2/Tetherin: a restriction factor of HIV-1.

Implication of the ubiquitination pathway in its antiviral activity

and countermeasures by the viral protein Vpu.

Nicolas Roy

3eme année de Doctorat

BioCIHP Meeting, Cochin Institute

23rd, April 2012

Laboratory director : Dr. Clarisse Berlioz-Torrent

Thesis 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

Entry

Reverse-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

Vif

Vpr 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

From Stenmark et coll, Nature, 2009

Decreased recycling of BST2

Endocytosis through AP-2

Recruitment by the ESCRT machinery

Lysosome

Late endosomes

MVB

Reduced cell surface level of BST2

Infected cells (Vpu+)

+ Vpu

Endosome

ESCRT

HRSUb

Increased rate of BST2 degradation

Janvier et al., PloS Pathogen, 2011

Janvier et al., Curr HIV. Res., 2012

BST2 is ubiquitinated and Vpu increases its ubiquitination

HA-Ub

Flag-BST2

Flag-BST2

HA-Ub + + + +- - - -

- - + + - - + +

Lysates Flag-IP

HA-Ub

Flag-BST2

Flag-BST2

HA-Ub

+ + + + + + + +

- -+ + - -+ +

NL4.3 W

T

NL4.3 U

del

NL4.3 W

T

NL4.3 U

del

Lysates Flag-IP

E3 ligases : siRNA screening

E1ATP

Ub 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

- siN

edd4

- siN

edd4

-L- s

iRNF1

89- s

iCBL

- siIT

CH- s

iWwp1

- siβ

-TrC

p

- siR

NF138

- siU

BAP1

BST2

Tubulin

siCD -

siNed

d4 -

siNed

d4-L

-siR

NF189

-

siCBL

-siI

TCH -siW

wp1 -

siRNF1

38 -

siβ-T

rCp -

siUBAP1

-

BST

2 ex

pres

sion

leve

l

(% o

f 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

- siN

edd4

- siN

edd4

-L- s

iRNF1

89- s

iCBL

- siIT

CH- s

iWwp1

- siβ-

TrCp

- siR

NF138

- siU

BAP1

β-TrCp2 -

- si β

-TrCp2

- siC

T

β-TrCp1 -

- siC

T

RNF189 -

- si R

NF189

- siC

T

- si β

-TrCp1

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

Effects of E3-ligases overexpression on BST2 distribution

pHA-E3 Ligases

J1

J3

J2

BST2

pCI-HA

BST2

BST2

Nedd4

BST2 Nedd4 BST2 RNF189 BST2 β-TrCp

β-TrCpRNF189

BST2 BST2

Immunofluorescence microscopy

HeLa

Redistribution of BST2 in an RNF189 positive compartment

Effect of E3-ligases overexpression on BST2 cell-surface expression level

RNF189 overexpression decreases BST2 cell-surface expression

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

- siN

edd4

- siN

edd4

-L- s

iRNF18

9- s

iCBL

- siIT

CH- s

iWwp1

- siβ-

TrCp

- 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

- siN

edd4

- siN

edd4

-L- s

iRNF1

89- s

iCBL

- siIT

CH- s

iWwp1

- siβ-

TrCp

- siC

T

- siC

T

- siN

edd4

- siN

edd4

-L- s

iRNF1

89- s

iCBL

- siIT

CH- s

iWwp1

- siβ-

TrCp

- 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

Tubulin

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

E1ATP

Ub 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

- NL4.3

WT

- NL4.3

Ude

l- N

L4.3 V

pu2.6

- NI

BST2

- gp160- SUgp120

- TMgp41

- Pr55GAG

- CAp24

- Tubulin

- Vpu

23 -

30 -

175 -

46 -

58 -

17 -

46 -

58 -

23 -

30 -

Mutations on Serine 52/56 (Vpu2.6) impairs Vpu-induced targeting of BST2 for degradation

Impaired interaction between Vpu and b-TrCp leads to stabilisation of an uncharacterized form of BST2

Role of b-TrCp on Vpu-induced downregulation of BST2- N

I - siN

edd4

- siN

edd4

-L- s

iRNF1

89- s

iCBL

- siC

D

- siN

edd4

- siN

edd4

-L- s

iRNF18

9- s

iCBL

- siC

D

NL4.3 WT NL4.3 Udel

BST2

- gp160- SUgp120

- TMgp41

- Pr55GAG

- CAp24

- Tubulin

23 -

30 -

175 -

46 -

58 -

23 -

46 -

- siIT

CH

- siW

wp1

- siβ-

TrCp

- siC

D

- NI - siIT

CH

- siW

wp1

- siβ-

TrCp

- siC

T

NL4.3 WT NL4.3 Udel

BST2

- gp160- SUgp120

- TMgp41

- Pr55GAG

- CAp24

- Tubulin

23 -

30 -

175 -

46 -

58 -

23 -

46 -

pHA-E3

pFlag-BST2

eGFP-Vpu

24h

Cell lysis

Flag pull-down

Western blot analysis

J1

J2

Effects of Vpu overexpression on BST2 binding with Nedd4, RNF189 and b-TrCp

Vpu connects BST2 to b-TrCp

Vpu does not interfere with the binding of BST2 with Nedd4 and RNF189

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:

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

Vpu connects BST2 to β-TrCp

Katy Janvier

Clarisse Berlioz-Torrent

Sophie Lambert

Stéphane Frémont

Grégory Pacini

Ursula Madjo

Sophie Abélanet

Vanessa Gourhand

Clarisse Berlioz-Torrent’s group

Platforms

Microscopy

Sequencing

Flow Cytometry

Stéphane Emiliani’s group

Effects of E3-ligases depletion on viral production

siRNA E3-ligasesViral

production

HIV-1 (NL4.3 VSV-G)

24h48h

HeLa

CAp24 quantification

WB

Infectivity

Neither Nedd4, RNF189 nor β-TrCp are required for HIV-1 release