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BACKGROUND • TFEB is a master gene expression regulator of the classical
autophagy-lysosome pathway. • Autophagy is a major self-degradative process in mammalian cells
with roles in cellular homeostasis. • Under basal conditions, TFEB is phosphorlyated by MTORC1,
resulting in the cytoplasmic retention of TFEB by 14-3-3 proteins. • When MTORC1 is inhibited, TFEB is dephosphorylated, prompting
its translocation to the nucleus where it increases the expression of genes encoding lysosomal proteins and the induction of autophagy.
• The conversion of LC3B-I to LC3B-II and its turnover is an indicator of autophagy induction and flux
• Sequestosome 1 (SQSTM1) binds LC3B-II and is degraded when autophagosomes fuse with lysosomes.
This work was supported by the NINDS (R01 NS084912),
the NIAID, NIH (AI084573), the IMPAACT Network and the California
HIV/AIDS Research Program (ID12-SD-255). We thank Olivier
Schwartz (Pasteur Institute) for pNL(AD8) and pNL(AD8)ΔNef.
HIV-1 infection induces TFEB dephosphorylation and nuclear localization
FIGURE 2 FIGURE 4 HIV-1 Nef inhibits TFEB nuclear translocation and autophagy
FIGURE 5 Beclin-1 is involved in TFEB nuclear translocation
HIV-1 Nef Inhibits Autophagy in Macrophages through Transcription Factor EB Sequestration
Grant R. Campbell1, Pratima Rawat1, Rachel S. Bruckman2, and Stephen A. Spector1,3
1Department of Pediatrics, Division of Infectious Diseases, and 2Division of Biological Sciences, University of California San Diego, La Jolla, California, USA, and 3Rady Children’s Hospital, San Diego, California, USA
University of California San Diego, Stein Clinical Research Building Room 432, 9500 Gilman Drive, La Jolla CA 92093-0672, USA [email protected] [email protected] Phone: +1 (858) 534 7477 Fax: +1 (858) 534 7411
FIGURE 1 HIV-1 infection induces autophagy
FIGURE 3 HIV-1 mediated autophagy is through TFEB
• HIV-1 infection induces autophagy in macrophages that is dependent upon the dephosphorylation and nuclear localization of TFEB
• HIV-1 Nef inhibits autophagy by binding BECN1, allowing TFEB to be phosphorylated by MTOR
Background: Transcription factor EB (TFEB) is a master gene expression regulator of the classical autophagy-lysosome pathway. Under basal conditions, TFEB is phosphorlyated by MTORC1, resulting in the cytoplasmic retention of TFEB through interactions with 14-3-3 proteins. When MTORC1 is inhibited, TFEB is dephosphorylated, prompting its translocation to the nucleus where it increases the expression of genes encoding lysosomal proteins. HIV-1 Nef acts as an anti-autophagic maturation factor through interaction with beclin-1, an essential autophagy and tumor suppressor protein. In this study, we investigated the role of Nef and TFEB in the modulation of autophagy during HIV-1-infection of human macrophages.
Methods: Monocyte-derived macrophages (MDM) were challenged with 105 TCID50 HIV-1Ba-L or HIV-1ΔNef per 5 x 105 cells. Autophagy and the role of TFEB was assessed using the expression, localization and phosphorylation of autophagy proteins combined with the lipidation of microtubule-associated protein 1 light chain 3B (LC3B) by immunoblotting, qRT-PCR, and fluorescence microscopy or by transducing MDM with shRNA for TFEB. Data were analyzed using the Mann-Whitney U test.
Results: Following exposure of macrophages to HIV-1Ba-L or heat-inactivated HIV-1, TFEB is dephosphorylated and translocated to the nucleus. This correlated with an increase in autophagy as evidenced by increased lipidation of LC3B, degradation of sequestosome and increased autophagosome formation (P < 0.01) and was attenuated by TFEB silencing (P < 0.05). In the presence of productive infection, TFEB phosphorylation and cytoplasmic sequestration started to increase and autophagy markers decrease by 5 days post-infection and by 7 days, levels were similar to the uninfected controls (P > 0.05). HIV-1ΔNef similarly induced the dephosphorylation and nuclear localization of TFEB that corresponded to an increase in autophagy during initial infection. Conversely, at 7 days post-infection nuclear accumulation of dephosphorylated TFEB and autophagy markers remained elevated (P < 0.02).
Conclusions: These results support a model whereby, during initial infection, the interaction between virion surface protein(s) and host receptors serve as a signal for autophagy initiation that is dependent upon the dephosphorylation and nuclear translocation of TFEB mediated by the inhibition of MTORC1. Once HIV-1 establishes a productive infection, Nef down regulates autophagy through the phosphorylation and cytosolic sequestration of TFEB. These findings help to explain how HIV-1 modulates autophagy to promote its own replication and cell survival, and further suggests that disrupting the autophagic balance within the infected cell can inhibit HIV-1 and potentially eliminate the infected cell.
RESULTS ABSTRACT
0 2 4 6 8
10
- + - + - + - + - +
1 3 5 7 10 Days
0
0.5
1
1.5
- + - + - + - + - +
1 3 5 7 10
CONCLUSIONS
60
12
kDa
0 5
10 15 20
- + - + - + - + - +
1 3 5 7 10
Fold
cha
nge
TF
EB
60
12
0 50
100 150
shC
ontro
l sh
BE
CN
1 sh
Con
trol
shB
EC
N1
0 10 Day
% B
EC
N1
rem
aini
ng
Macrophages were infected with 0.01 MOI HIV-1Ba-L. Top, cells were harvested, lysed and fractionated into nuclear and
cytoplasmic fractions at 1, 3, 5, 7 and 10 days post-infection. The localization of TFEB was analyzed using Western blot. Bottom,
cells were fixed, permeabilized, and stained with antibody to TFEB (red), lysosomal-associated membrane protein 1 (LAMP1)
(green), and DAPI (blue). Early HIV-1 infection induces TFEB dephosphorylation (as indicated by its faster migration in SDS-
PAGE) and nuclear translocation (white colocalization with DAPI) suggesting that the TFEB signaling pathway is activated. At
later time points TFEB is again sequestered within the cytoplasm. * P < 0.05; n = 4.
0
2
4
6
- + - + - + - + - +
1 3 5 7 10
Nuc
lear
TFE
B
TFEB
ACTB
60
40
kDa HIV-1 infection
Days post-infection 0
0.5 1
1.5 2
2.5
- + - + - + - + - +
1 3 5 7 10
Cyt
osol
ic T
FEB
TFEB
Histone H3
60
12
kDa
Cytoplasm Nucleus
* * * * *
Days post-infection TFEB
LAMP1 DAPI
1 3 6 9
HIV
-1 u
ninf
ecte
d H
IV-1
infe
cted
Macrophages were infected with 0.01 MOI HIV-1Ba-L. Cells were harvested and lysed at 1, 3, 5, 7 and 10 days post-infection.
LC3B lipidation and SQSTM1 degradation were analyzed by Western blot. HIV-1 infection upregulates autophagy at 1 and 3 d
post-infection and is down regulated by 5 d and is the same as mock-infected controls by 7 d. * P < 0.05; n = 6.
Macrophages were infected with HIV-1Ba-L (+), mock infected (-) or with heat-inactivated HIV-1Ba-L (H) and harvested at 24 and
72 h post-treatment. Heat inactivated HIV-1Ba-L increased LC3B lipidation and SQSTM1 degradation at 24 h indicating that active
HIV-1 infection is not required for the initial induction of autophagy. * P < 0.05; n = 4.
72 - - + H - H +
24 -4
ACTB
SQSTM1
LC3-I LC3-II
HIV-1 infection Hours post-infection
60
12
40
kDa
0
0.5
1
1.5
- - H + - H +
-4 24 72
SQ
STM
1
0 5
10 15 20 25 30
- - H + - H +
-4 24 72
LC3B
-II:L
C3B
-I
HIV-1
Hours
HIV-1
Hours
0
0.5
1
1.5
- + - + - + - + - +
1 3 5 7 10
SQ
STM
1
* *
*
0
10
20
30
40
- + - + - + - + - +
1 3 5 7 10
LC3B
-II:L
C3B
-I * *
*
- - + - + + 3 5 7 10
- + - + 1
ACTB
SQSTM1
LC3-I LC3-II
HIV-1 infection Days post-infection
60
12
40
kDa
HIV-1
Days
HIV-1
Days
* *
*
*
* * *
*
Nucleus
TFEB
ACTB
60
40
kDa
0 0.5
1 1.5
2
- + - + - + - + - +
1 3 5 7 10
Fold
cha
nge
TF
EB
TFEB
Histone H3
Cytoplasm
* * *
TFEB
ACTB
60
40 BE
CN
1 R
NA
i C
ontro
l R
NA
i
TFEB
Histone H3
Days post-infection
HIV-1 infection
Macrophages silenced for beclin-1 (BECN1) were infected with
HIV-1Ba-L. Cells were harvested, lysed and fractionated into nuclear and
cytoplasmic fractions. The localization of TFEB was analyzed using
Western blot. BECN1 silencing resulted in the abrogation of TFEB
dephosphorylation and nuclear localization post-HIV-infection. * P <
0.05; n = 4.
BECN1
ACTB
* * Control RNAi BECN1 RNAi
*
* *
Macrophages were infected with HIV-1NL(AD8)ΔNef or HIV-1NL(AD8)ΔNef. Top, cells were harvested, lysed and fractionated into
nuclear and cytoplasmic fractions over time and the localization of TFEB analyzed by Western blot.. Bottom, the degradation of
SQSTM1 and LC3B lipidation were analyzed by Western blot. HIV-1 Nef is required for HIV-1 induced inhibition of TFEB
nuclear translocation and induction of autophagy at late time points. * P < 0.05, n = 4.
ACTB
SQSTM1
LC3-I LC3-II
60
12
40
kDa HIV-1 infection
Days - +
1 - +
3 - +
5 - +
7 10 - +
60
12
40
kDa
ACTB
SQSTM1
LC3-I LC3-II
0 10 20 30 40 50
- + - + - + - + - +
1 3 5 7 10
HIV-1
Days
LC3B
-II:L
C3B
-I
0 0.5
1 1.5
2
- + - + - + - + - +
1 3 5 7 10
SQ
STM
1
HIV-1
LC3B
-II:L
C3B
-I
SQ
STM
1
HIV
-1N
L(AD
8)ΔN
ef
HIV
-1N
L(AD
8)
* *
* * *
* * * *
* * *
* * *
TFEB
ACTB
60
40
kDa
0 0.5
1 1.5
2 2.5
3
- + - + - + - + - +
1 3 5 7 10
Fold
cha
nge
TF
EB
TFEB
Histone H3
60
12
kDa
Cytoplasm Nucleus
* *
*
TFEB
ACTB
60
40
HIV
-1N
L(AD
8)ΔN
ef
HIV
-1N
L(AD
8)
0 2 4 6 8
- + - + - + - + - +
1 3 5 7 10
Fold
cha
nge
TF
EB
TFEB
Histone H3
60
12
Days post-infection
HIV-1 infection
HIV-1NL(AD8) HIV-1NL(AD8)ΔNef
* * * *
* *
* * * * * *
Macrophages silenced for TFEB were infected with HIV-1Ba-L. The
degradation of SQSTM1 and LC3B lipidation over time were analyzed
using Western blot. TFEB is required for HIV-1 induced autophagy
induction with little to no LC3B lipidation or SQSTM1 degradation
observed in TFEB silenced cells. * P < 0.05, n = 4.
Control RNAi
- - + - + + 3 5 7 10
- + - + 1
ACTB
SQSTM1
LC3-I LC3-II
HIV-1 infection Days post-infection
60
12
40
kDa
0 2 4 6 8
10
- + - + - + - + - +
1 3 5 7 10
LC3B
-II:L
C3B
-I * *
*
HIV-1
Days
0
0.5
1
1.5
- + - + - + - + - +
1 3 5 7 10
SQ
STM
1 * * *
HIV-1
Days
- - + + 5 7 10
- +
60
12
40
kDa + -
3 - +
1
ACTB
SQSTM1
LC3-I LC3-II
HIV-1 infection Days post-infection
0
0.5
1
1.5
- + - + - + - + - +
1 3 5 7 10
SQ
STM
1
0 2 4 6 8
10
- + - + - + - + - +
1 3 5 7 10
LC3B
-II:L
C3B
-I
HIV-1
Days
HIV-1
Days
TFEB RNAi
TFEB
ACTB
0 50
100 150
shC
ontro
l sh
TFE
B
shC
ontro
l sh
TFE
B
0 10 Day
% T
FEB
re
mai
ning
* *
ACKNOWLEDGEMENTS
Basal conditions HIV infection
Day 10 0 0 10 shTFEB - - + +
shControl + + - - Day shBECN1 shControl
10 - +
0 - +
0 + -
10 + -
