HIV-1 Nef Dimerization is Important for AP2 Recruitment and CD4 … · 2020. 6. 6. · •...
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HIV-1 Nef Dimerization is Important for AP2 Recruitment and CD4 Downregulation Sherry T. Shu, Lori A. Emert-Sedlak and Thomas E. Smithgall Background: The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef hijacks host cell trafficking pathways to downregulate membrane-bound receptors, including CD4 via the adaptor protein 2 (AP2) complex. CD4 downregulation is linked to enhanced viral infectivity and immune escape, identifying this important Nef function as a rational target for new antiretroviral drug discovery. Methods: We developed a cell-based bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with the AP2 complex and CD4. Interacting protein pairs were fused to complementary, non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with CD4 or AP2 result in complementation of YFP and a bright fluorescent signal by confocal microcopy. Results: Nef interaction with both CD4 and the AP2 α and σ2 subunits was readily visualized in live cells by BiFC assay. Co-expression of the AP2 α subunit enhanced the Nef:AP2 σ2 BiFC signal and vice versa, suggesting that the AP2 α/σ2 hemi-complex interacts cooperatively with Nef. Mutagenesis of Nef residues R134, E174, and D175, which stabilize the Nef docking surface for AP2 in a recent co-crystal structure (PDB: 4NEE), substantially reduced AP2 interaction without affecting CD4 binding. Remarkably, a dimerization-defective mutant of Nef failed to interact with either CD4 or AP2 in the BiFC assay, indicating that the quaternary structure of Nef is required for CD4 and AP2 recruitment as well as CD4 downregulation. Small molecule inhibitors of Nef dimerization also reduced Nef interactions with AP2 and CD4 by BiFC, providing further support for this idea and identifying the Nef dimer interface as a target for inhibitor action. Conclusions: Here we describe a fluorescence complementation assay to visualize interactions between Nef, CD4 and the AP2 complex in live cells. Using this assay, we observed that the AP2 α and σ2 subunits interact cooperatively with Nef, and that the interaction is dependent upon an internal Nef salt bridge present in the recent crystal structure of the Nef:AP2 complex. Complex assembly also requires an intact Nef dimerization interface, consistent with previous observations that dimerization-defective Nef mutants fail to downregulate CD4. Small molecules that inhibit Nef dimerization also suppress AP2 and CD4 recruitment, which may provide a mechanistic explanation for their effects on HIV infectivity. Abstract Summary and future directions Inhibition of Nef dimerization disrupts Nef:CD4:AP2α-σ2 complex Visualizing Nef interactions with CD4 and AP2 using Bimolecular Fluorescence Complementation (BiFC) Introduction Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA Ren et al., eLife 2014 • BiFC enables study of the interaction between two proteins in a cell-based assay. The bright YFP variant Venus was used in these experiments. • Nef interacts with CD4, AP2α, and AP2σ2 in 293T cells by BiFC. • BiFC reveals interactions of Nef with CD4 and AP2 in cells without disrupting their normal functions. • Interaction of Nef-VN-HA:AP2α-V5-VC increases in the presence of AP2σ2 in transfected 293T cells. • Mutations at Nef internal salt bridge decrease Nef binding to AP2α and σ2 subunits but Nef interaction with CD4 remains intact. • Reagents and technical support: Drs. Jerrod Poe, John Jeff Alvarado, Carolyn Coyne , Linton Traub, and Gary Thomas. • Grants: NIH K01 OD018431 (PI: STS) and R01 AI057083 (PI: TES) Acknowledgements • We demonstrate the interactions between Nef, CD4 and AP2 subunits in 293T cells using BiFC. AP2α and σ2 subunits interact cooperatively with Nef in the cell, consistent with previous reports that they bind as a hemi-complex. • Mutations and small molecule inhibitors targeting the Nef dimerization interface indirectly disrupt the interaction between Nef and the AP2, indicating the importance of Nef dimerization in complex assembly. • To study the temporal aspects of Nef:AP2 interaction, we will use live-cell video microscopy to investigate the effect of mutations and inhibitors at binding interface on the rate of complex assembly and trafficking. • To investigate the role of Nef dimerization and AP2 binding in vivo, we will infect humanized mice with HIV-1 Nef dimerization deficient mutants, as well as AP2 and CD4 binding deficient mutants. BLT (Blood-Liver-Thymus) humanized mice have been developed in our laboratory. • We will continue testing the efficacy of Nef inhibitors that disrupt Nef dimerization and CD4 downregulation in vitro and in vivo for new drug development. Nef:AP2α-σ2 Nef intramolecular hydrogen bonds including Nef D175:R134 Model of membrane- anchored Nef:CD4:AP2 complex α σ2 Nef loop L 164 L 165 α σ2 D 174 D 175 R 134 Q 104 CD4-VC CD4 V5 VC AP2α V5 VC AP2α-VC AP2σ2 V5 VC AP2σ2-VC Nef VN HA Nef-VN Nef-VC Nef V5 VC BiFC Nef-VN Partner-VC Partner Nef Partner Nef CD4-VC Partner Nef BiFC AP2σ2-VC AP2α-VC Merge Nef-VN + BiFC fusion proteins CD4 alone + AP2α and AP2σ2 + Nef + AP2α, AP2σ2 and Nef 0 0.1 0.5 1.5 BiFC Nef AP2α (μg): BiFC: Nef-VN + AP2σ2-VC AP2σ2 + - - - + + - - 0.1 + + - - 0.5 + + - - 1.5 + - - + - + - 0.1 + - + - 0.5 + - + - 1.5 + - + AP2α-VC AP2α (μg) AP2σ2-VC AP2σ2 (μg) Nef-VN Nef-VN WT R134E ED-AA α-VC σ2-VC CD4-VC R134E ED-AA Nef-VN: DMSO B9 (3 μM) Nef CD4 α σ2 Nef CD4 α σ2 20 40 60 80 100 % of DMSO HIV-1 Nef interactions with CD4 and the AP2 complex • The HIV accessory protein Nef is a virulence factor required for high titer viral replication, immune escape, and AIDS progression. • Nef interacts with CD4 and induces endocytosis through the Adaptor Protein 2 (AP2) complex. Nef dimerization may play an important role in its interaction with AP2. • Structural basis of Nef interactions with CD4 and the AP2 complex. B Nef:AP2 (4NEE) + Nef dimer (4U5W) σ2 α Nef (4NEE) Nef dimer (4U5W) VN: VC: Nef-WT Nef-WT Nef-4D Nef-4D Nef-4D AP-2α Nef-4D AP-σ2 Nef-4D CD4 I109 L112 Y115 F121 Y115 F121 L112 I109 • A dimerization-defective mutant of Nef (4D) fails to interact with CD4, AP2α, and AP2σ2 in 293T cells and can no longer down-regulate CD4. • B9 is a small molecule Nef inhibitor predicted to bind to the Nef dimer interface by Autodock Vina. B9 Interferes with Nef dimerization in the BiFC assay. • B9 disrupts Nef to interact with CD4 and AP2 α and σ2 subunits, and inhibits CD4 down regulation in infected cells. + α and σ2 CD4 alone + Nef + Nef-4D + Nef-R134E PDB:1EFN Fluorescence intensity of cell surface CD4 determined by flow cytometry Non-BiFC constructs + - + - + - - + - + - + - + - + - + + - + - + - σ2-VC α-VC σ2 α Nef-VN WT R134E ED-AA AP2 1.00 0.75 0.50 0.25 0.00 Nef:AP2 Interaction BiFC/IF Ratio AP2 α-σ2 hemi-complex interacts cooperatively with Nef to downregulate cell-surface CD4 BiFC:IF Ratio 0.0 0.5 1.0 1.5 Nef-4D mutant replaces I109, L112, Y115 and F121 with Asp. The significant lost of interaction suggests that the quaternary structure of Nef is important for effector recruitment and function Nef 2 Nef 1 B9 The predicted B9 binding site is away from the AP2 interaction site, suggesting an allosteric inhibitory effect of B9. Docking model of B9 bound to the Nef dimer (PDB: 1EFN). BiFC: IF ratio compared to DMSO control Surface expression of CD4 on HIV-infected cells in the presence of B9 CD4 surface expression (% control) 20 40 60 80 100 0 0 uninfected WT dNef B9 (3μM)
Transcript of HIV-1 Nef Dimerization is Important for AP2 Recruitment and CD4 … · 2020. 6. 6. · •...
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HIV-1 Nef Dimerization is Important for AP2 Recruitment and CD4 Downregulation
Sherry T. Shu, Lori A. Emert-Sedlak and Thomas E. Smithgall
Background: The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef hijacks host cell trafficking pathways to downregulate membrane-bound receptors, including CD4 via the adaptor protein 2 (AP2) complex. CD4 downregulation is linked to enhanced viral infectivity and immune escape, identifying this important Nef function as a rational target for new antiretroviral drug discovery.Methods: We developed a cell-based bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with the AP2 complex and CD4. Interacting protein pairs were fused to complementary, non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with CD4 or AP2 result in complementation of YFP and a bright fluorescent signal by confocal microcopy. Results: Nef interaction with both CD4 and the AP2 α and σ2 subunits was readily visualized in live cells by BiFC assay. Co-expression of the AP2 α subunit enhanced the Nef:AP2 σ2 BiFC signal and vice versa, suggesting that the AP2 α/σ2 hemi-complex interacts cooperatively with Nef. Mutagenesis of Nef residues R134, E174, and D175, which stabilize the Nef docking surface for AP2 in a recent co-crystal structure (PDB: 4NEE), substantially reduced AP2 interaction without affecting CD4 binding. Remarkably, a dimerization-defective mutant of Nef failed to interact with either CD4 or AP2 in the BiFC assay, indicating that the quaternary structure of Nef is required for CD4 and AP2 recruitment as well as CD4 downregulation. Small molecule inhibitors of Nef dimerization also reduced Nef interactions with AP2 and CD4 by BiFC, providing further support for this idea and identifying the Nef dimer interface as a target for inhibitor action. Conclusions: Here we describe a fluorescence complementation assay to visualize interactions between Nef, CD4 and the AP2 complex in live cells. Using this assay, we observed that the AP2 α and σ2 subunits interact cooperatively with Nef, and that the interaction is dependent upon an internal Nef salt bridge present in the recent crystal structure of the Nef:AP2 complex. Complex assembly also requires an intact Nef dimerization interface, consistent with previous observations that dimerization-defective Nef mutants fail to downregulate CD4. Small molecules that inhibit Nef dimerization also suppress AP2 and CD4 recruitment, which may provide a mechanistic explanation for their effects on HIV infectivity.
Abstract
Summary and future directions
Inhibition of Nef dimerization disrupts Nef:CD4:AP2α-σ2 complex
Visualizing Nef interactions with CD4 and AP2 using Bimolecular Fluorescence
Complementation (BiFC)
Introduction
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh PA
Ren et al., eLife 2014
• BiFC enables study of the interaction between two proteins in a cell-based assay. The bright YFP variant Venus was used in these experiments.
• Nef interacts with CD4, AP2α, and AP2σ2 in 293T cells by BiFC.
• BiFC reveals interactions of Nef with CD4 and AP2 in cells without disrupting their normal functions.
• Interaction of Nef-VN-HA:AP2α-V5-VC increases in the presence of AP2σ2 in transfected 293T cells.
• Mutations at Nef internal salt bridge decrease Nef binding to AP2α and σ2 subunits but Nef interaction with CD4 remains intact.
• Reagents and technical support: Drs. Jerrod Poe, John Jeff Alvarado, Carolyn Coyne , Linton Traub, and Gary Thomas.
• Grants: NIH K01 OD018431 (PI: STS) and R01 AI057083 (PI: TES)
Acknowledgements
• We demonstrate the interactions between Nef, CD4 and AP2 subunits in 293T cells using BiFC. AP2α and σ2 subunits interact cooperatively with Nef in the cell, consistent with previous reports that they bind as a hemi-complex.
• Mutations and small molecule inhibitors targeting the Nef dimerization interface indirectly disrupt the interaction between Nef and the AP2, indicating the importance of Nef dimerization in complex assembly.
• To study the temporal aspects of Nef:AP2 interaction, we will use live-cell video microscopy to investigate the effect of mutations and inhibitors at binding interface on the rate of complex assembly and trafficking.
• To investigate the role of Nef dimerization and AP2 binding in vivo, we will infect humanized mice with HIV-1 Nef dimerization deficient mutants, as well as AP2 and CD4 binding deficient mutants. BLT (Blood-Liver-Thymus) humanized mice have been developed in our laboratory.
• We will continue testing the efficacy of Nef inhibitors that disrupt Nef dimerization and CD4 downregulation in vitro and in vivo for new drug development.
Nef:AP2α-σ2 Nef intramolecular hydrogen bonds including Nef D175:R134
Model of membrane-anchored Nef:CD4:AP2 complex
ασ2
NefloopL164
L165 ασ2
D174
D175
R134
Q104
CD4-VC CD4 V5 VC
AP2α V5 VCAP2α-VC
AP2σ2 V5 VCAP2σ2-VC
Nef VN HANef-VN
Nef-VC Nef V5 VC
BiFCNef-VN Partner-VC
PartnerNef PartnerNef
CD
4-VC
PartnerNefBiFC
AP2σ
2-VC
AP2α
-VC
Merge
Nef
-VN
+
BiFC fusion proteins
CD4 alone+ AP2α and AP2σ2+ Nef+ AP2α, AP2σ2 and Nef
0 0.1 0.5 1.5
BiF
CN
ef
AP2α (μg):BiFC: Nef-VN + AP2σ2-VC
AP2
σ2
0 . 0
0 . 5
1 . 0
1 . 5
BiF
C:I
F R
ati
o
+---+
+--
0.1+
+--
0.5+
+--
1.5+
--+-+
-0.1+-+
-0.5+-+
-1.5+-+
AP2α-VCAP2α (μg)AP2σ2-VCAP2σ2 (μg)Nef-VN
Nef
-VN
WT
R13
4EED
-AA
α-VC σ2-VC
CD4-VC
R134E ED-AANef-VN:
DMSO B9 (3 μM)
Nef
CD4
α
σ2
Nef CD4 α σ2
20
40
60
80
100
% o
f DM
SO
HIV-1 Nef interactions with CD4 and the AP2 complex• The HIV accessory protein Nef is a virulence factor required for high titer viral
replication, immune escape, and AIDS progression.
• Nef interacts with CD4 and induces endocytosis through the Adaptor Protein 2 (AP2) complex. Nef dimerization may play an important role in its interaction with AP2.
• Structural basis of Nef interactions with CD4 and the AP2 complex.
B
Nef:AP2 (4NEE) + Nef dimer (4U5W)
σ2
α
Nef (4NEE)Nef dimer (4U5W)
VN:VC:
Nef-WTNef-WT
Nef-4DNef-4D
Nef-4DAP-2α
Nef-4DAP-σ2
Nef-4DCD4
I109
L112
Y115
F121
Y115F121
L112
I109
• A dimerization-defective mutant of Nef (4D) fails to interact with CD4, AP2α, and AP2σ2 in 293T cells and can no longer down-regulate CD4.
• B9 is a small molecule Nef inhibitor predicted to bind to the Nef dimer interface by Autodock Vina. B9 Interferes with Nef dimerization in the BiFC assay.
• B9 disrupts Nef to interact with CD4 and AP2 α and σ2 subunits, and inhibits CD4 down regulation in infected cells.
+ α and σ2CD4 alone+ Nef+ Nef-4D+ Nef-R134E
PDB:1EFN
Fluorescence intensity of cell surface CD4 determined by flow cytometry
Non-BiFC constructs
+ - + - + -- + - + - +
0 . 0 0
0 . 2 5
0 . 5 0
0 . 7 5
1 . 0 0
Ne
f:A
P-2
In
tera
cti
on
BiF
C/I
F R
ati
o
- + - + - ++ - + - + -
σ2-VC
α-VC
σ2
α
Nef-VN
WT R134E ED-AA
AP2
1.00
0.75
0.50
0.25
0.00Nef
:AP2
Inte
ract
ion
BiFC
/IF R
atio
AP2 α-σ2 hemi-complex interacts cooperatively with Nef to downregulate
cell-surface CD4
BiFC
:IFR
atio
0.0
0.5
1.0
1.5
Nef-4D mutant replaces I109, L112, Y115 and F121 with Asp. The significant lost of interaction suggests that the quaternary structure of Nef is important for effector recruitment and function
Nef2Nef1
B9The predicted B9 binding site is away from the AP2 interaction site, suggesting an allosteric inhibitory effect of B9.
Docking model of B9 bound
to the Nef dimer (PDB: 1EFN).
BiFC: IF ratio compared to DMSO control
Surface expression of CD4 on HIV-infected cells
in the presence of B9
CD
4 su
rface
exp
ress
ion
(% c
ontro
l)
20
40
60
80
100
0
0
uninfected WT dNef B9 (3μM)
