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