1
1
2
Supplemental figures
3
Supplemental Fig. 1. Autophagy is inhibited by Vif after 11 days of infection (a) H9 cells
4
were infected by NL4.3WT or NL4.3ΔVif for 11 days at a MOI of 1. Then, cells were fixed
5
and autophagy was analyzed as described in Fig.4. (b) Chloroquine was added to infected H9
6
cells for 3h before cell fixation. Scale bars = 10 µm.
7
8
2
Supplemental figure 1
% of autophagic cells
H9 cells
NL4.3WT
NL4.3∆Vif
50
*
NL4.3 WT
NL4.3ΔVif
40
30
20
10
0
Dapi
LC3
p24
Overlay
H9 cells + chloroquine
% of autophagic cells
80
NL4.3WT
NL4.3∆Vif
60
40
20
0
Dapi
9
10
11
LC3
p24
Overlay
***
NL4.3 WT
NL4.3ΔVif
3
12
Supplemental Fig.2. Vif inhibits autophagy in HIV-infected Jurkat cells and in HIV-
13
transfected HEK cells. (a) LC3-II expression was analyzed in Jurkat cells infected by
14
NL4.3WT or NL4.3ΔVif for 3 days at a MOI of 1. Fold induction was calculated as the
15
intensity of the LC3-II immunoblot obtained in cells infected by NL4.3WT compared to cells
16
infected by NL4.3ΔVif. Values were normalized to those obtained with anti-GAPDH Ab. (b)
17
HEK cells were transfected with the molecular clone pNL4.3WT or pNL4.3ΔVif for 24 h.
18
LC3-II expression was analyzed by western blotting. Fold induction was calculated as the
19
intensity of the LC3-II immunoblot obtained in cells transfected by pNL4.3WT compared to
20
cells transfected by pNL4.3ΔVif. Values were normalized to those obtained with anti-
21
GAPDH Ab. Data are representative of at least 5 independent experiments.
22
4
Supplemental Figure 2
(a)
(b)
LC3-II
α-GAPDH
α-GAPDH
α-Vif
α-Vif
LC3-II/GAPDH
23
24
25
LC3-I
LC3-II
α-LC3
α-LC3
1 2.7
LC3-II/GAPDH
1
1.7
5
26
Supplemental Fig. 3. Vif inhibits autophagy induced by drugs when ectopically
27
expressed in HEK cells. (a) HEK cells were transfected with the plasmid expressing
28
mCherryVif. One day later, 20 µM MG132 and 2 µM Torin were added for 4 h. Cells were
29
then fixed, and endogenous LC3 was labeled using the anti-LC3 Ab as described in the
30
section Materials and Methods. Results are from at least 2 independent blind experiments. (b)
31
Autophagy was induced by starvation (EBSS for 3h) in HEK cells transfected with either a
32
plasmid expressing Flag or Flag-Vif, in presence or absence of anti-proteases (pepstatin A and
33
Ed64 at 10 µg/mL). LC3-II expression was analyzed by western blotting and was normalized
34
to the GAPDH expression level. Data are representative of 3 independent experiments.
35
6
Supplemental Figure 3
(a)
% of autophagic cells
70
Dapi
LC3
mCherryVif
Overlay
50
40
30
20
10
0
Vif
(b)
**
*
2.5
pepstatinA
+E64d
α-LC3
α-GAPDH
α-Vif
- +
- +
LC3-I
LC3-II
LC3-II/GAPDH (AU)
3xFlag 3xFlag-Vif
***
60
-
+
3xFlag-Vif
3XFlag
2
1.5
1
0.5
pepstatinA
+E64d
0
- +
- +
36
37
38
Supplemental Fig. 4. Vif increases intracellular p24 expression independently of the
39
presence of A3G. (a) HEK-CD4-CXCR4 cells were transfected with the same amount of
7
40
pNL4.3WT or pNL4.3ΔVif. 24h later, these cells were co-cultured for 3 days with Jurkat
41
cells. The percentage of infected CD4 T cells was analyzed by FACS. (b) p24 was measured
42
in lysates of Jurkat cells infected as described in (a) using a p24 antigen capture ELISA
43
(Beckman Coulter). (c) SupT1cells infected by co-culture as described in (a) were enriched by
44
limiting dilution cloning and expression of p55Gag and p24 proteins was analyzed by
45
Western blotting using the anti-Gag Ab.
46
8
Supplemental Figure 4
% of infected cells
60
50
(b)
NL4.3 WT
NL4.3ΔVif
8000
P24 (µg/mL)
(a)
40
30
20
NL4.3 WT
NL4.3ΔVif
6000
4000
2000
10
0
0
(c)
LC3-I
α-LC3
LC3-II
α-GAPDH
α-Vif
p55
α-p24
p24
LC3-II/GAPDH
1
2.8
p55/GAPDH
1
0.3
47
48
49
Supplemental Fig.5. LC3 cannot bind to the SOCS box-like motif when the EloB/C
50
complex is bound to Vif. (a) Binding prediction of LC3 (blue) on Vif (grey, red and
9
51
magenta) obtained by rigid docking as shown in Figure 2c. (b) Crystal structure of the
52
complex between Vif and EloB/C (both Elongin chains are shown in orange and Cullin 5
53
chains are depicted in tan ribbon).
54
55
10
Supplemental Figure 5
(a)
(b)
EloB/C
Vif-1-172
Vif-1-172
G120
LC3
N-ter
C-ter
N-ter
56
57
C-ter