Molecular Events Underlying Cell-Associated HIV Transmission
Rahm GummuluruBoston University School of Medicine
Department of Microbiology
HIV – Dendritic Cell Interactions• Robust virus replication in dendritic cell (DC) – T cell co-cultures
(Cameron et al, 1992).• Mature DCs can transfer HIV-1 particles to CD4+ T cells at an
enhanced efficiency– primarily because of establishment of stable DC – T cell
conjugates (Macdonald et al, 2003)
– Putative evasion from neutralizing antibody responses (Sagar etal, 2012)
DC – T cell Virological SynapseDC – T cell Immunological Synapse
MHC – TCR (CD3)CD4 or CD8ICAM1 – LFA1CD80/86 – CD28
DC T cell
HIV
EJI 2005 35:1741 MHC – TCR (CD3)CD4 or CD8ICAM1 – LFA1 CD80/86 – CD28 CD81
Dendritic Cells Facilitate HIV-1 Infection!
• DC – T cell co-cultures are sites of robust viral replication#
Kinetics of Establishment of Productive Infection in CD4+ T cells is Enhanced in the Setting of DC – T cell Co-cultures#
T cells alone#
DC - T transfer (50% fusion) = 38.6 ± 1.75 min T cells alone (50% fusion) = 84 ± 2.5 min
Magnitude of Virus Replication in #CD4+ T cells is Enhanced in the
Setting of DC – T cell Co-cultures #
DC – T cell #T cells alone#DC – T cell#
J. Virol. 2002 76: 10692 - 71"
0 25 50 75 100 1250
20
40
60
80
100
Time (min)
DCs alone#
Dendritic Cells: Hostile Cellular Environment for HIV
CD4CCR5
Produc've Infec'on
Reverse Transcrip1on
X Fusion
& EntryUncoa1ng
Nuclear ImportX Mx2
Integra1on
Transcrip1on
Assembly & Release
Viral proteinsynthesis
Lysosome PRRs
Virus Par'cle Degrada'on Virus Sensing
An'gen presenta'on
Receptor‐ ‐‐mediated endocytosis
C‐‐‐type lec'n receptors
Phagocytosis
Innate Responses
Scavenger receptors
Intrinsic Responses
Cytoplasmic Sensor of Gag (?) X
SamHD1 Apobec3G
X
Dendritic Cell-Associated HIV-1 trans Infection
MVB
HIV-attachment factors
CD4+ T cells
Concentration within Membrane Invaginations
CD4+ T cells
Captured HIV-1 Particles transferred to T cells Without de novo virus replication in Dendritic Cells
Dendritic Cell-Associated HIV-1 trans Infection
MVB
HIV-attachment factors
CD4+ T cells
Concentration within Membrane Invaginations
CD4+ T cells
Captured HIV-1 Particles transferred to T cells Without de novo virus replication in Dendritic Cells
HIV-1 Attachment factors on Dendritic Cells
Envelope Glycoprotein gp120 - Dependent Binding
•C-type lectin receptors- Recognition of the glycosylated gp120- DC-SIGN, Langerin, DCIR
•Heparan sulfate proteoglycans (Syndecans)- Polyanionic surface
- Charge-based interactions with gp120
Geijtenbeek et al, Cell 2000; Turville et al, Nat. Immunol 2002; de Witte et al, PNAS 2007; Lambert et al, Blood 2008;
Envelope Glycoprotein-Independent Capture of HIV-1 Particles by Dendritic Cells
Env‐‐‐deficient virus par'cles derived from divergent cellular sources remain competent for capture by dendri'c cells (J. Virol. 2009 83: 3496‐‐‐3506 )
Immature DCs Mature DCs0
5
10
15
20
25HEK293T cell‐‐‐derived virus
0
3
6
9
12
15Jurkat T cell‐‐‐derived virus
Immature DCs Mature DCs
0
1
24
3
4CD4+ T cell‐‐‐derived virus
Immature DCs Mature DCs
Macrophage‐‐‐drived virus
HIV‐‐‐1 (wt)
HIV‐ ‐‐1/Δenv
0
2
6
8
1 0
e rtup aCgag
p2 4%
Immature DCs Mature DCs
HIV‐‐‐1 (wt)
HIV‐ ‐‐1/Δenv
Host cellular determinants incorporated in virus particle membrane might play a role in HIV capture by dendritic cells.
~ 7 ‐‐‐ 14 Env trimers per par1cle
Zhu et al., Nature 2006Sougrat et al., PLoS Pathogens 2007
Lipid Rafts
Binding
Budding
Fusion
Modified from progenics.com/hiv.cfm
galactoyslceramide sphingomyelin cholesterol
proteinsglycosphingolipid
Lipid Rafts
HIV Incorporates Molecules from Host Membrane
Modified from Higashi, Glycoworld
•Lipid and protein composition of the virus particle membrane is determined by thesite of virus assembly and exit.
•Virus particles enriched in cholesterol, sphingolipids, glycosphingolipids, and lipidraft associated cellular proteins such as CD59 and tetraspanin proteins.
•Strategy to identify HIV determinant necessary for capture by DCs• Selective depletion of virus particle membrane constituents
Cholesterol Sphingomyelin Glycosphingolipids
HIV LipidomeHIV Proteome
TetraspaninsGPI-linked proteins Class II MHC
GSLdeficient
Hatch, J.Virol 2009
Removal of GSLs from the virion
1009080706050403020100
mock
hat is the HIV par'cle determinant necessary for DC cVirus Particle-Associated Proteins are not
W aptuRequired for DC Mediated HIV-1 Capture
re?
Izquierdo-Useros, Blood 2009
Removal of Protein from the virion
mock Pronase treated
Virus Particle Associated Glycosphingolipids a Essential for HIV-1 Capture by DCs
re
Maturation of DCs Enhances Env-Independent Glycosphingolipid Mechanism of HIV-1 Capture
100 101 102 103 104FL1-H
Immature DCs
Mature DCs
Gag‐‐‐eGFP VLP Capture
0
25
50
75
100
MA
HIV wt‐‐‐Gag‐‐‐eGFP
p2 p1myr
Hatch et al, J. Virol. 2009 83: 3496‐‐‐3506; Izquierdo‐ ‐‐Useros et al, Blood 2009 113:2732‐‐‐2741
HIV VLPs produced in the presence of PDMP are impaired for capture by mature DCs
GSL Biosynthesis PathwayPDMPCeramide X Glucosylceramide Glycosphingolipids
GalactosylceramideSphingomyelin
dihydroceramide
CTxB IP
Gag‐ ‐‐eGFP
MA CA NC p6 eGFP
Glycosphingolipids (GSLs): Raft-Resident Lipids•Lipid por'on of GSL, ceramide, is embedded in the outerleaflet of the plasma membrane, and their glycan moie'es project into the extracellular space.
GSL Biosynthesis Pathway
0.72 (+/-0.15) 0.37 (+/-0.07)***
GM3/p24 (+/-SD)
0.33 (+/-0.15*) **
******
NT
Galactosylceramide Sphingomyelin
UGCG ST3GalV
GM3-Depleted Virions are Deficient for Capture by Mature DCs
Puryear et al, PNAS 2012 109: 7475 – 7480
GSL Biosynthesis Pathway
Ceramide Glucosylceramide LactosylceramidedihydroceramideUGCG
GM3ST3GalV
GM3
FB1
CERA MIDE
SPHINGOMYELIN(major mb phospholipid)
SULFATIDES,GM4
GLYCOSPHINGOLIPIDSLactosyl Ceramide
Blood group: lactoseries and neolactoseries
Gangliosides(sialic-acid-containing GSLs )
globosides
Glucosyl transferase / GCS
PDMP
Galactosyl transferase / GalCer synthase
Sphingomyelin synthase
A4GALT
Glucosyl Ceramide
Lactosyl transferase
ST3GAL5 B4GALT
Lac3Cer
SPHINGOLIPIDS
B3GNT5
CERTER-golgi transport
GSL Biosynthesis Pathwaydihydroceramide
FAPP2ER-golgi transpor t
XGM3a-series
GD3b-series
GT3c-series
asialo-gsls
GA2 Gb3
B4GALT B4GALT B4GALT
GM2 GD2 GT2 GA1 GM2α Gb4 GalGb3Cer
GM1 GD1 GT1
others others others others others others others
‐‐‐ 150nm (~ virus sized) par1cles‐‐‐ cholesterol coupled to TopFluor dye
Mock (PS) +Lipid
Liposome system for analyzing HIV Capture by Mature DCs
HIV
Composi1on~ 45% cholesterol>30% phospholipids~10% PS0.5‐‐‐3.5% GM3
Composi1on 45% cholesterol* 54% DPPC1% PS
Composi1on 45% cholesterol* 53% DPPC1% PS1% Lipid
Mock (PS)
+Lipid
NeuAcGal
Glc
NeuAcGal Glc
GalNAc
GalNeuAc
NeuAc
Gal
ceramide GalCer GM3 (α2,3-sialic acid) GQ1b (α2,8-sialic acid)
Inclusion of GM3 in Virus-sized Liposomes Results in Enhanced Mature DC capture
PS Cer Gal GM3 GQ1b0
100
200
300
Type of liposome
p = 0.0002***
GFP
Blocking GM3 Results in Attenuated Capture of HIV by Mature Dendritic Cells
Competition With Liposomes
GFP
Competition With α-GM3 Fab fragments
Immune Activation and GM3 Enrichment in HIV-1 Particles
• GM3 is the major ganglioside in the plasmamembrane of lymphocytes and monocytes
• GM3 synthase activity is upregulated– upon immune activation, and– upon initiation of monocyte to macrophage
differentiation
100 101 102 103FL1-H
104
enricheduntreatedmock
GM3 enrichment in Virus Producer Cells Produces HIV that is Captured by DCs with Enhanced Efficiency
100 101 102 103
GM3(Alexa488)
enriched
untreated
mock
GM3 Expression on Virus Producer Cells
10 0 10 1 10 102 3 10 4
FL1-HGM3(Alexa488)
untreated
enriched
mock
HeLa Monocyte Macs Monocyte/Pam3 CysK4
GM3(Alexa488)
mock +GM3
Enrichment
0.0
0.5
2.03
1.5
21.0
NTC +Pam3CysK40
1
THP Treatment (HIVLai)
HIV-1 Capture by Dendritic Cells
Summary:GM3 mediates HIV capture by DCs
Puryear et al, PNAS 2012 109: 7475 – 7480
Loss of Function
- Knockdown of GM3 synthase results in decreased capture
HIV
Gain of Function
-Enrichment with GM3 (immune activation or lipid addition) results in enhanced capture
-Liposomes with GM3 have enhanced capture
HIV Liposome
Competition Assays
- GM3 liposomes compete for capture
- α-GM3 Fab blocks capture
What is the DC‐‐‐receptor responsible for GM3‐‐‐dependent HIV‐‐‐1 capture?
Characteristics of the GM3-Dependent HIV Capture Mechanism on Dendritic Cells
4. α2,3‐‐‐Sialic Acid Requirement
** **
Neuraminidase Treatment of HIV
1. Pronase Sensi1ve
Mock Pronase DC pretreatment
0
25
50
75
100
125
2. IFN‐‐‐αInducible
None (immature DCs) Pam3CysK4 Poly(I:C)LPS IFN!TNF!
0
100
200
300
400
500
600
700
Dendritic Cell Stimulation
3. Myeloid Cell Specific
0
1
2
3
4
5
NT+ IFNα
SIGLEC Family of Lectins
• Sialic acid-recognizing Ig-superfamily lectins• Major homologous family of I-type lectins
divided into 2 sub-groups– Evolutionary conserved sub-group (Siglecs-1, -2
and -4)– Rapidly evolving CD33 and Siglec-3-related sub-
group (Siglecs-3 and -5-13 in primates)• Broadly expressed in cells of the immune
system
cDCs
Siglec1 (CD169)•expressed exclusively on myeloid cells•expression induced by type I IFN•binds to terminal α2,3-linked sialic acid residues
SIALIC-ACID-BINDING IMMUNOGLOBULIN-LIKE LECTINs
0
25000
50000
75000
1000000
500000
0
1500000
1000002000000
125000
150000 p=0.0093500000
3000000
2500000
Maturation of Dendritic Cells Enhances CD169 Expression
Quantitative FACS HIV-1 Capture
0
10
20
30
40
Monocyte Day 2 Day 4 Immature DCs (day 6) Mature DCs (LPS)
CD169
No StainIsotype Control Scrambled shRNA CD169 shRNA
DC-SIGN shRNA
DC-SIGN CD169 CD86
• Lentivector/shRNA-mediated knockdown of CD169 or DC-SIGN in mature DCs
Selective Depletion of CD169 in Mature Dendritic Cells Abrogates HIV-1 Capture
Immature Mature0
20
40
60
80
100
120
Immature Mature0
20
40
60
80
100
120
140
160
180CD169 DC-SIGNCD169/DC-SIGNIsotype (IgG1) Isotype (IgG2b) Isotype (both) Non-treated
Virus Binding HIV-1 trans Infection
Neutralizing Antibodies Against CD169 block DC Mediated HIV-1 Capture & Trans Infection
Exogenous Expression of CD169 in Receptor-Naïve Cells Rescues GSL-Dependent HIV-1 Capture & Trans Infection
• Express human CD169 in Raji B cell line(non-permissive for HIV capture)
• Incubate with WT virus (NT), virusdeficient in GSL (PDMP), or Env-deficient
virus• Co-culture with T cells
100 101
CD16 9
102
FL1- H
103 1040
20
40
60
80
100 Raji Isotype Raji/CD169
0
1
2
3
4
5
6
NT
PDMP
0
5000
10000
15000
20000
25000
30000
Raji Raji/CD169
NT
PDMP
HIV-1 Trans Infection
Capture and transfer of GSL-deficient HIV-1 particles by Raji/CD169 cells is attenuated
HIV-1 Capture
Raji Raji/CD1690
2
4
6
8
Raji Raji/CD169
HIV/LaiHIV/LaiΔenv
HIV-1 Capture
CD169
actin
0.1 1 10Volume (µl)
1000.0
0.2
0.4
0.6
0.8
1.0
1.2
Blank
GM3Raji
Raji/CD169
GalCer GM3 GM1 GQ1b
p=0.003
p=0.018
0
50
100
150
200
250Liposome Capture Assay Liposome Compe11on Assay
GM3 Recognition by CD169 is Essential for HIV-1 Capture
WT = human CD169R96A = no affect on sialic acid bindingR116A = abrogates sialic acid binding
Vinson M et al, JBC 1996
96
116
Lack of HIV-1 Capture by Cells Expressing CD169 Mutant Deficient for Sialic Acid Recognition
Unstained Isotype WT R96A R116A
CD169
Cell Surface CD169 Expression
CD169
actin
A
B
HIV‐‐‐1 Capture
****
D
0
10
20
30***
0
10
20
30Mock CD169 R96A R116A
C
Murine CD169 can Mediate RetroviralCapture and Trans Infection
ne CD169 expressio o seen on m s
• 72%id80 e
ntity between h type I transm
similarity in th
ical betwee nd mouse
hum40 an a
nd pr
llu
mM oo uck se CD16980- bo
he
6t 0
40
•H i
g s20
t0
emb30 ran e
oteMuirnines CD169
e ex1 t0 race lar domain
• 17 Ig like0d10om1a0 in1s0 (N10-terminal NVT-set dGoSmL-/-ain and 16 CN-Tset doGmSLa-/-ins)
CD169• Sialic acid binding site is i1 d000 e00 nt
HIV-1 Transfer n human a MLV TransferCD1691000000
800000
• GM3 gangliosides are enr6 i0 c00 h0 ed inlipi
d bilayer of60 s000 i0 mple retr s, MLVoviruJ. Viro
400000 Chan et al, l 2008
2 3 4 5
100Unstained IsotypeMurine CD169
0
200000
0 20
40
60
100
20
50
Mock Murine CD169
0
20000
40000
80000
•Muri Murine CD169 Expression n als HIV-1 Capture yeloid cell MLV Capture
Mock
Murine CD169
Mock Murine CD169
0.02 (+/-0.02) 0.45 (+/-0.18) 0.70 (+/-0.17)
HIV Gag-mCherry VLPs Co-localize with CD169 on Mature DC Surface and at the DC – T cell Virological Synapse
No VLP VLP < 10min VLP 120minDC : T cell
0.84 (+/-0.10)Correlation Coefficient
Deconvolution Microscopy
HIV CD169
500 nmZ
X
ZX
X Y X
Super-resolution Microscopy
CD169#HIV#
MDC#
CD169#HIV#
MDC#
Localization of HIV-1 Particles in CD169+ Plasma Membrane Invaginations of Myeloid DCs
HIV-1 Particles Captured by CD169 are Trafficked to Non-Lysosomal Compartments#
HIV-1 Evasion of Adaptive Immune Responses
All Surface0.0
1.0
0.8
0.6
0.4
0.2
VRC01PermeabilizedSurface
HIV VRCO1
All Surface0.0
0.8
0.6
0.4
0.2
1.0
CD169
HIV-1 CD169
HIV VRCO1
PermeabilizedSurface
HIV-1 CD169
Conclusions
• Expression of CD169 on monocyte-derived DCs, inflammatory DCs andblood myeloid DCs is required for GM3-dependent HIV-1 capture.
• Blocking CD169 with neutralizing antibody or depletion via shRNA results inloss of DC-dependent HIV-1 capture and trans infection.
• Induced expression of CD169 in a non-permissive cell confers the ability tocapture and transfer virus. Mutation that eliminates sialic acid binding (R116A) results in
attenuation of virus capture by CD169.
• HIV-bound CD169 re-localizes to a polarized region of DC and is co-localized with HIV-1 particles at the DC – T cell infectious synapse.
• HIV-1 particles captured by CD169 are trafficked to plasma membraneinvaginations Invariably accessed by surface-applied anti-gp120 monoclonal antibodies
Puryear & Akiyama et al, PLoS Path 2013 Izquierdo-Useros et al, PLoS Biology 2012
DC-‐SIGN
CD4 & CoR
GSLs
Syndecan endocytosis
infecPon Env de
pend
ent
GM3 de
pend
ent
degradaPon
Trans infecPon
?
?
de novo virus
blocked
CD169
Virus producer cell
T cell
Dendri9c cell
Immune Ac1va1on GM3 Incorpora'on
in Virus Par1cles Immune Ac1va1on DC-‐mediated HIV trans infec1on
CD169+ DC Mediated HIV-1 Trans Infection!• Dependent on a host cell-‐derived ligand (GM3) and receptor (CD169)• Parasi1za1on of cell-‐to-‐cell recogni1on mechanism by HIV for virus dissemina1on• Protec1on from innate and adap1ve immune responses
Acknowledgements
Boston University Chemistry & Photonics
Björn ReinhardXinwei Yu Amin
Feizpour
Vutara, IncManasa Gudheti
Wendy Puryear
Hisashi Akiyama Nora Ramirez Suzanne Geer Caitlin Miller
Past Members Steve Hatch Hiren
Patel Erica Li
Funding: NIH RO1-AI064099, R56-AI104393
University of PittsburghZandrea Ambrose
Boston University School of MedicineManish Sagar Deborah Anderson
Tsuneya Ikezu
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